Use of an anti-ang2 antibody

ABSTRACT

A method of preventing or treating cancer using an anti-Ang2 antibody or an antigen-binding fragment thereof that specifically binds to an angiogenesis-inducing factor Angiopoietin-2 (Ang2) and complexes to a Tie2 receptor while bound with Ang2.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Korean Patent Application No.10-2013-0112089 filed on Sep. 17, 2013 in the Korean IntellectualProperty Office, the entire disclosure of which is hereby incorporatedby reference.

INCORPORATION BY REFERENCE OF ELECTRONICALLY SUBMITTED MATERIALS

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted herewith and identifiedas follows: 11,702 bytes ASCII (Text) file named “717998_ST25.TXT,”created Jul. 29, 2014

BACKGROUND

1. Field

Provided is an anti-Ang2 antibody or an antigen-binding fragment thereofthat specifically binds to an angiogenesis-inducing factorAngiopoietin-2 (Ang2) and complexes with a Tie2 receptor while bound toAng2, and a method for enhancing an efficacy of an anticancer agent, anda use thereof for combination therapy for treating a cancer.

2. Description of the Related Art

Angiopoietin-1 (Ang1) and Angiopoietin-2 (Ang2) bind to Tie2 receptorspecifically expressed in vascular endothelial cells, thereby performingcomplicated functions in angiogenesis and vascular remodeling.

Angiopoietin-1 (Ang1) is a ligand which is thought to activate the Tie2receptor, relate to the formation and maturation of blood vessels, andcontribute to vascular stabilization by increasing interaction betweenvascular endothelial cells and pericytes. In addition, it is believedthat Ang1 functions as a key regulator for maintaining the stabilizationof blood vessels by maintaining the barrier function of vascularendothelial cells. The vascular endothelial cells are activated in thestatus of the overexpression of VEGF or inflammation, and vascularpermeability is increased. Ang1 induces the stabilization of vascularendothelial cells and reduces vascular permeability by accelerating thejunctional integrity of the vascular endothelial cells.

The overexpression of Ang2 in a variety of solid cancers and bloodcancers has been reported, and Ang2 expression in a serum often servesas a biomarker of poor prognosis of anticancer therapy. Theoverexpression of Ang2 has been reported not only in the cancers butalso in various diseases including sepsis, bacterial infection, lunginjury, kidney injury, etc. Such overexpressed Ang2 is thought toinhibit stabilization of vascular endothelial cells by competing withAng1. Due to such correlation with such pathological conditions, Ang2has been considered to be a target in therapeutic intervention.Currently, a variety of drugs targeting Ang2 are currently in theclinical or preclinical stages of development, and they have a varietyof forms including a peptibody, bispecific antibody, monoclonalantibody, etc.

However, most of the Ang2-target drugs prevent Ang2 from binding to theTie2 receptor.

SUMMARY

One embodiment provides an anti-Ang2 antibody or an antigen-bindingfragment thereof, wherein the anti-Ang2 antibody specifically binds toan angiogenesis-inducing factor Ang2 (Angiopoietin-2) and complexes witha Tie2 receptor while bound to Ang2 to induce the activation of the Tie2receptor.

Another embodiment provides a hybridoma cell line producing a monoclonalantibody of the anti-Ang2 antibody.

Another embodiment provides an anti-Ang2 antibody-Ang2 complex whereinthe anti-Ang2 antibody or an antigen-binding fragment thereof and Ang2are bound to each other.

Another embodiment provides a method of activating Tie2 receptorincluding administering the anti-Ang2 antibody or an antigen-bindingfragment thereof to a subject in need thereof.

Another embodiment provides a method of inhibiting angiogenesisincluding administering the anti-Ang2 antibody or an antigen-bindingfragment thereof to a subject in need thereof.

Another embodiment provides a method of decreasing vascular permeabilityincluding administering the anti-Ang2 antibody or an antigen-bindingfragment thereof to a subject in need thereof.

Another embodiment provides a method of inducing normal blood vesselformation including administering the anti-Ang2 antibody or anantigen-binding fragment thereof to a subject in need thereof.

Another embodiment provides a method of preventing and/or treating adisease related to angiogenesis, increase of vascular permeability,and/or decrease of normal blood vessel formation, includingadministering the anti-Ang2 antibody or an antigen-binding fragmentthereof to a subject in need thereof.

Another embodiment provides an adjuvant for enhancing an anticanceragent, wherein the adjuvant includes the anti-Ang2 antibody or anantigen-binding fragment thereof as an active ingredient.

Another embodiment provides a method of enhancing an anticancer agentincluding administering the anti-Ang2 antibody or an antigen-bindingfragment thereof together with an anticancer agent to a subject in needthereof.

Another embodiment provides a method of combination therapy forpreventing and/or treating a cancer, including administering andanticancer agent and the anti-Ang2 antibody or an antigen-bindingfragment thereof to a subject in need thereof.

Another embodiment provides a method of inhibiting Ang2 and/oractivating a Tie2 receptor including administering the anti-Ang2antibody or an antigen-binding fragment thereof to a subject.

Another embodiment provides a method of diagnosing a disease related tooverexpression of Ang2 using the anti-Ang2 antibody or anantigen-binding fragment thereof.

Another embodiment provides a complex in which the anti-Ang2 antibodyand the antigen-binding fragment thereof, Ang2, and a Tie2 receptor arebound to one another (e.g., the anti-Ang2 antibody and theantigen-binding fragment thereof specifically binds Ang2, and Ang2 bindsa Tie2 receptor).

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a graph of the Ang2-Tie2 competition ELISA results showing thesuppression of binding between Tie2 receptor and Ang2 according to thetreatment concentration of an anti-Ang2 antibody.

FIG. 2A is an annotated gel photograph of the immunoblotting resultsshowing the level of Tie2 receptor and a phosphorylated Tie2 receptoraccording to the concentration of an anti-Ang2 antibody.

FIG. 2B is an annotated gel photograph of the immunoblotting resultsshowing the level of Tie2 receptor and a phosphorylated Tie2 receptoraccording to the concentration of an anti-Ang2 antibody compared to thatof a control antibody (RG).

FIG. 2C is a graph showing a ratio of phosphorylated Tyr in Tie2receptor.

FIG. 3A is an annotated gel photograph of the immunoblotting resultsshowing the phosphorylation of proteins involved in the downstreamsignaling of a Tie2 receptor by an anti-Ang2 antibody.

FIG. 3B is an annotated gel photograph of the immunoblotting resultsshowing the phosphorylation of Tie2 receptor and a protein (Akt)involved in the downstream signaling of a Tie2 receptor by an anti-Ang2antibody.

FIG. 3C is a graph showing the results of 3B as numerical values.

FIG. 4 is a graph showing the ELISA results showing the formation of acomplex by the binding of an anti-Ang2 antibody with Ang2 and Tie2.

FIG. 5 is an annotated gel photograph of the immunoblotting resultsshowing the phosphorylation of a Tie2 receptor of a monomeric anti-Ang2antibody and proteins involved in the downstream signaling of the Tie2receptor.

FIGS. 6A and 6B provide a series of fluorescent images showing Ang1 andan anti-Ang2 antibody lead to similar intracellular internalization of aTie2 receptor.

FIGS. 7A and 7B include graphs showing the proliferation of vascular(7A) and lymphatic endothelial cells (7B) when treated with an anti-Ang2antibody.

FIG. 8 is a graph showing the migration of lymphatic endothelial cellswhen treated with an anti-Ang2 antibody.

FIG. 9 is a graph showing inhibition of cell permeability induced by TNFor LPS when treated with Ang1 or Ang2 antibody.

FIG. 10 is a graph showing the mRNA expression of inflammation-relatedcell adhesion substances (ICAM-1, E-selectin) induced by LPS stimulationwhen treated with Ang1 or Ang2 antibody.

FIG. 11 is a graph showing the strengths of fluorescence obtained frominflammatory cells when treated with an anti-Ang2 antibody as relativefolds, and it shows the number of the inflammatory cells adhered tovascular endothelial cells.

FIG. 12 is a graph showing tumor volume in a Colo205 tumor model whentreated with an anti-Ang2 antibody.

FIG. 13 is a graph showing survival rate of LLC tumor model when treatedwith an anti-Ang2 antibody.

FIG. 14 are fluorescent images showing blood vessels and pericytes ofLLC tumor model when treated with an anti-Ang2 antibody throughimmunostaining, indicating the structural normalization of blood vesselsby treatment of the anti-Ang2 antibody.

FIG. 15A are fluorescent images showing blood vessels and pericytes ofLLC tumor model when treated with an anti-Ang2 antibody through lectinperfusion, indicating the functional normalization of blood vessel bytreatment of the anti-Ang2 antibody.

FIG. 15B is a graph obtained by quantification of the result of lectinperfusion in LLC tumor model treated with an anti-Ang2 antibody.

FIG. 16 are images showing the necrotic area of tumor cells in a LCCtumor model when treated with an anti-Ang2 antibody.

FIG. 17 is a fluorescent image showing lymph nodes when treated with ananti-Ang2 antibody.

FIG. 18A is a graph showing tumor volume changes in a lung cancer animalmodel when co-treated with cisplatin and an anti-Ang2 antibody.

FIG. 18B are fluorescent images showing transportation of cisplatin toinside of tumor cells in a lung cancer animal model when co-treated withcisplatin and an anti-Ang2 antibody.

FIG. 18C are images showing the necrotic area of tumor cell whenco-treated with cisplatin and an anti-Ang2 antibody (upper portion); anda graph showing ratio of necrosis/total sectional area (%) (lowerportion).

FIG. 19A is a graph showing weight changes of normal mice when treatedwith an anti-Ang2 antibody.

FIG. 19B are fluorescent images showing blood vessels of normal micewhen treated with an anti-Ang2 antibody.

DETAILED DESCRIPTION

The inventors have verified that an antibody which specifically binds toAng2 but does not inhibit binding between Ang2 and a Tie2 receptor andforms a complex (antibody/Ang2/Tie2) together with Ang2 and the Tie2receptor has a characteristic of inducing the dimerization of theantibody Through this, it can induce the activation of the Tie2 receptorand its downstream signaling by effectively clustering the Tie2 receptorin the complex. The antibody having this mechanism of action inhibitsAng2 and has a dual function of Ang2 neutralization (inhibition ofangiogenesis) and the normalization of blood vessels; that is, theantibody binds to Ang2 to induce the intracellular internalization anddegradation thereof, thereby lowering the level of circulating Ang2, andat the same time, it induces Tie2 downstream signaling by binding to aTie2 receptor together with (via) Ang2 to activate the Tie2 receptor,similarly to Ang1, and induces the normalization of blood vessels. Thenormalization of blood vessels may mean that abnormally formed bloodvessels in cancer cells are converted into a structurally andfunctionally normal status; that is, the blood vessels in cancer cellsbecome possessing normal structure and having decreased vascularpermeability, and recover their normal function performance abilities.

Provided is a therapeutic antibody targeting an angiogenesis-inducingfactor Ang2 and particularly, to an anti-Ang2 antibody which not onlyinhibits the functions of Ang2 by specifically binding to Ang2 therebyinhibiting angiogenesis and decreasing density of blood vessels in tumortissue but also induces the activation of Tie2 by allowing Ang2 to bindTie2, thereby structurally and/or functionally normalizing the bloodvessels. The anti-Ang2 antibody may bind Ang2 in such a way that Ang2may still bind with Tie2. The anti-Ang2 antibody may not directly bindto Tie2 receptor, but it can form a complex with Tie2 by binding Ang2which, in turn, binds Tie2 receptor. The anti-Ang2 antibody has theeffects of treating diseases by binding to a Tie2 receptor together withAng2 to activate the Tie2 receptor and thus induce thestructural/functional normalization of blood vessels, along with thedown-regulation of Ang2 in diseases related to the dysfunction and theabnormal activation of blood vessels such as cancer, sepsis, eyedisorders, etc.

In addition, the anti-Ang2 antibody, or an antigen-binding fragmentthereof, has the effect of both inhibiting angiogenesis by Ang2neutralization and normalizing blood vessels. When co-administered withvarious anticancer agents, the anti-Ang2 antibody or an antigen-bindingfragment thereof, increases the transporting efficiency of theco-administered anticancer agents into cancer cells or cancer tissues,and thus, it can be useful as an adjuvant (efficacy enhancing agent) orsensitizer for increasing the effects of the anticancer agents. The term“normalizing the blood vessel” may refer to normalize the blood vesselsin tumor by converting the tumor vessels having structural features suchas wide diameter, a lot of branches, a lot of sprouts, being tortuous,and/or few pericytes (green in FIG. 15; wherein pericytes (green) arefew and far from the vessels), and functional features such as poorperfusion, into normal vessels having normal structures and functions.

One embodiment provides an anti-Ang2 antibody or an antigen-bindingfragment thereof, specifically binding to (recognizing) anangiogenesis-inducing factor Ang2 (Angiopoietin-2) and binding to a Tie2receptor together with Ang2 (via Ang2). Thus, the anti-Ang2 antibody oran antigen-binding fragment thereof may specifically recognize and/orbind to Ang2 and bind to Tie2 receptor via Ang2. Also, the anti-Ang2antibody or an antigen-binding fragment thereof may induce theactivation of the Tie2 receptor. Such activation of Tie2 receptor may beinduced by an increase in the phosphorylation of Tie2 receptor and/orthe phosphorylation of proteins related to the downstream signal pathwaythereof, for example, at least one selected from the group consisting ofAkt (NM_(—)005163), eNOS (NM_(—)000603), 42/44 (NM_(—)002745), etc.Also, the anti-Ang2 antibody or an antigen-binding fragment thereof mayinduce the intracellular internalization of a Tie2 receptor. In otherwords, the anti-Ang2 antibody or an antigen-binding fragment thereof maybind to Ang2 and the Tie2 receptor via Ang2 to form a complex and inducethe activation of the Tie2 receptor, by not inhibiting binding betweenAng2 and the Tie2 receptor while specifically binding to Ang2, unlikethe pre-existing anti-Ang2 antibodies. Therefore, the anti-Ang2 antibodyor an antigen-binding fragment thereof may increase the phosphorylationof a protein related to the downstream signal pathway of Tie2 receptor,such as at least one selected from the group consisting of Akt, eNOS,and 42/44, compared to the case using (treating) no antibody or anyanti-Ang2 antibody inhibiting the binding between Ang2 and Tie2receptor, such as antibody 4H10 (SEQ ID NOs: 12 & 13), RG antibody(Regeneron Co.), etc.

The Ang2 protein which functions as an antigen against the antibody isclosely related to angiogenesis, and as a soluble ligand present inblood, it is widely involved in angiogenesis, metastasis, cancer cellinvasion, etc. The Ang2 may be derived from mammals including primatessuch as humans and monkeys and rodents such as rats and mice and forexample, it may be human Ang2 (e.g., NCBI Accession No. 015123, etc.),monkey Ang2 (e.g., NCBI Accession No. Q8MIK6, etc.), mouse Ang2 (e.g.,NCBI Accession No. 035608, etc.), and rat Ang2 (e.g., NCBI Accession No.035462, etc.), but is not limited thereto.

The Tie2 receptor (TEK tyrosine kinase), which is an Angiopoietin-1receptor, is expressed in vascular endothelial cells in various mammalssuch as mouse (NM_(—)013690; NP_(—)038718), rat, and human(NM_(—)000459; NP_(—)000450), and is involved in various downstreamsignaling.

As explained above, the anti-Ang2 antibody or an antigen-bindingfragment thereof is characterized in that the antibody whichspecifically binds to Ang2 but does not inhibit binding between Ang2 andTie2 receptor and forms a complex (antibody/Ang2/Tie2) together withAng2 and the Tie2 receptor, has a characteristic of inducing thedimerization of the antibody, and through this, it can induce theactivation of the Tie2 receptor and its downstream signaling byeffectively clustering the Tie2 receptor which constitutes the complex.By virtue of such an action mechanism, the anti-Ang2 antibody and theantigen-binding fragment thereof inhibits Ang2 functions by binding toAng2 to induce the intracellular internalization and degradation thereofand thus lowers the level of circulating Ang2 and at the same time, itinduces Tie2 downstream signaling by binding to the Tie2 receptortogether with Ang2 to activate the Tie2 receptor, like Ang1 and inducesthe stabilization of vascular endothelial cells. By having such dualfunctions, the antibody and the antigen-binding fragment thereof can beusefully employed to treat not only symptoms (disorders) due to theoverexpression of Ang2 but also symptoms (disorders) due to the decreasein the stabilization of vascular endothelial cells, that is, theincrease of vascular penetration.

In addition, the anti-Ang2 antibody or an antigen-binding fragmentthereof normalizes blood vessels, thereby facilitating transportation ofa pharmaceutical agent to lesions (e.g., Ang2 expressing cancer tissues)and increasing sensitiveness to the pharmaceutical agent, to enhance thetherapeutic efficacy of the pharmaceutical agent and achieve desiredeffects with a decreased dose of the pharmaceutical agent. Therefore,the anti-Ang2 antibody or an antigen-binding fragment thereof may beused as an adjuvant for enhancing the efficacy of an existingpharmaceutical agent, e.g., an existing anticancer agent, byco-administration with the pharmaceutical agent.

The anti-Ang2 antibody or an antigen-binding fragment thereof mayrecognize all or part (for example, at least one amino acid selectedfrom the group consisting of the amino acid residue site exposed to theoutside of loop) of loop 1 (of SEQ ID NO: 11, a site from 417^(th) aminoacid to 434^(th) amino acid) of human Ang2 (hAng2; SEQ ID NO: 11;Accession # O15123) or an amino acid sequence site including 2 to 20, 2to 15, 2 to 10, or 2 to 5 contiguous amino acids including at least oneamino acid residue exposed to the outside of loop 1 of SEQ ID NO: 11 asan epitope, or specifically bind to this site.

Ang2 (SEQ ID NO: 11) MWQIVFFTLS CDLVLAAAYN NFRKSMDSIG KKQYQVQHGSCSYTFLLPEM DNCRSSSSPY VSNAVQRDAP LEYDDSVQRLQVLENIMENN TQWLMKLENY IQDNMKKEMV EIQQNAVQNQTAVMIEIGTN LLNQTAEQTR KLTDVEAQVL NQTTRLELQLLEHSLSTNKL EKQILDQTSE INKLQDKNSF LEKKVLAMEDKHIIQLQSIK EEKDQLQVLV SKQNSIIEEL EKKIVTATVNNSVLQKQQHD LMETVNNLLT MMSTSNSAKD PTVAKEEQISFRDCAEVFKS GHTTNGIYTL TFPNSTEEIK AYCDMEAGGGGWTIIQRRED GSVDFQRTWK EYKVGFGNPS GEYWLGNEFVSQLTNQQRYV LKIHLKDWEG NEAYSLYEHF YLSSEELNYRIHLKGLTGTA GKISSISQPG NDFSTKDGDN DKCICKCSQMLTGGWWFDAC GPSNLNGMYY PQRQNTNKFN GIKWYYWKGS GYSLKATTMM IRPADF

For example, the anti-Ang2 antibody may recognize Q418, P419, acombination of Q418 and P419 positioned at loop 1 of SEQ ID NO: 11, oran amino acid sequence site including 2 to 20, 2 to 15, 2 to 10, or 2 to5 contiguous amino acids including the amino acid residue of Q418, P419,or combination of Q418 and P419 of SEQ ID NO: 11 as an epitope, orspecifically bind to this site. In one embodiment, the anti-Ang2antibody may recognize the amino acid residues of Q418 and P419 of SEQID NO: 11 as an epitope, or specifically bind to this portion.

Q418, P419, or an amino acid site including them, to which the anti-Ang2antibody specifically binds, is an exposed amino acid residue positionedat loop 1 of the three dimensional structure of Ang2, and it isconsidered to directly participate in binding between Ang2 and Tie2receptor or to be a site regulating it.

In Q418, P419, or an amino acid site including them, to which theanti-Ang2 antibody specifically binds, the term “contiguous amino acid”may refer to amino acids which are consecutive on primary structure, butalso amino acids which are adjacent to one another on the primary,secondary, or tertiary structure of a protein.

As not only the anti-Ang2 antibody recognizing and/or specificallybinding to the above site but also an antibody or an antigen-bindingfragment thereof which competes with the anti-Ang2 antibody for bindingcan inhibit Ang2 and at the same time, form a complex with Ang2 and Tie2receptor (i.e., by binding of antibody-Ang2 to the Tie2 receptor) toactivate Tie2. This competitively-binding antibody may be an antibodyrecognizing a site adjacent to the aforementioned site on its threedimensional structure as an epitope and/or a specific binding site. Thecompetitively-binding antibody may have a binding affinity with Ang2 of0.1 pM to 50 nM, particularly 1 pM to 30 nM, 2 pM to 20 nM, or 1 nM to10 nM.

Therefore, the anti-Ang2 antibody or an antigen-binding fragment thereofmay be at least one selected from the group consisting of an antibody oran antigen-binding fragment thereof recognizing the aforementioned siteas an epitope or specifically binding thereto.

In a particular embodiment, the anti-Ang2 antibody or an antigen-bindingfragment thereof may comprise or consist essentially of:

at least one heavy chain complementarity determining region (CDR)selected from the group consisting of a polypeptide (CDR-H1) includingthe amino acid sequence of SEQ ID NO: 1, a polypeptide (CDR-H2)including the amino acid sequence of SEQ ID NO: 2, and a polypeptide(CDR-H3) including the amino acid sequence of SEQ ID NO: 3, or a heavychain variable region including the at least one heavy chaincomplementarity determining region;

at least one light chain complementarity determining region selectedfrom the group consisting of a polypeptide (CDR-L1) including the aminoacid sequence of SEQ ID NO: 4, a polypeptide (CDR-L2) including theamino acid sequence of SEQ ID NO: 5, and a polypeptide (CDR-L3)including the amino acid sequence of SEQ ID NO: 6, or a light chainvariable region including the at least one light chain complementaritydetermining region;

a combination of said at least one heavy chain complementaritydetermining region and said at least one light chain complementaritydetermining region; or

a combination of the heavy chain variable region and the light chainvariable region.

More particularly, the anti-Ang2 antibody or an antigen-binding fragmentthereof may comprise or consist essentially of:

a heavy chain complementarity determining region including a polypeptide(CDR-H1) including the amino acid sequence of SEQ ID NO: 1, apolypeptide (CDR-H2) including the amino acid sequence of SEQ ID NO: 2,and a polypeptide (CDR-H3) including the amino acid sequence of SEQ IDNO: 3, or a heavy chain variable region including the at least one heavychain complementarity determining region; and

a light chain complementarity determining region including a polypeptide(CDR-L1) including the amino acid sequence of SEQ ID NO: 4, apolypeptide (CDR-L2) including the amino acid sequence of SEQ ID NO: 5,and a polypeptide (CDR-L3) including the amino acid sequence of SEQ IDNO: 6, or a light chain variable region including the at least one lightchain complementarity determining region.

In particular, the heavy chain complementarity determining region of theanti-Ang2 antibody or an antigen-binding fragment thereof may have aminoacid sequences, for example, as set forth in the following Table 1.

TABLE 1 Heavy Chain CDR Amino Acid Sequences CDRH1-KABAT CDRH2-KABATCDRH3-KABAT SDYAWN YINYSGNTDYNPSLKS GNFEGAMDY (SEQ ID NO: 1)(SEQ ID NO: 2) (SEQ ID NO: 3)

Likewise, the light chain complementarity determining region of theanti-Ang2 antibody or an antigen-binding fragment thereof may have aminoacid sequences, for example, as set forth in the following Table 2.

TABLE 2 Light Chain CDR Amino Acid Sequences CDRL1-KABAT CDRL2-KABATCDRL3-KABAT KASQSVSNDVA YASNRYP QQDYSSPWT (SEQ ID NO: 4) (SEQ ID NO: 5)(SEQ ID NO: 6)

In one embodiment, the heavy chain variable region of the antibody orthe antigen-binding fragment thereof may include the amino acid sequenceof SEQ ID NO: 7:

(SEQ ID NO: 7) DVQLQESGPGLVKPSQSLSLTCTVTGYSIT SDYAWN WIRQFPGNKLEWMGYINYSGNTDYNPSLKS RSSITRDTSKNQFFLQLNSVTTGDTATYYCAR GN FEGAMDY WGQGTSVTVSS

(In SEQ ID NO: 7 above, the underlined bold letters are CDRH1, CDRH2,and CDRH3 in sequence)

The light chain of the antibody according to one embodiment may includethe amino acid sequence of SEQ ID NO: 9.

(SEQ ID NO: 9) SIVMTQTPKFLLVSAGDRVTITC KASQSVSNDVA WYQQKPGQSPKLLIY YASNRYP GVPDRFTGSGYGTDFTFTISTVQAEDLAVYFC QQDYSSPWT FGG GTKLEIK

(In SEQ ID NO: 9 above, the underlined bold letters are CDRL1, CDRL2,and CDRL3 in sequence)

In this regard, the anti-Ang2 antibody or an antigen-binding fragmentthereof may include a heavy chain variable region including the aminoacid sequence of SEQ ID NO: 7, a light chain variable region includingthe amino acid sequence of SEQ ID NO: 9, or a combination of the heavychain variable region and the light chain variable region.

For example, the anti-Ang2 antibody or an antigen-binding fragmentthereof may include a heavy chain variable region including the aminoacid sequence of SEQ ID NO: 7 and a light chain variable regionincluding the amino acid sequence of SEQ ID NO: 9.

In one embodiment, the Anti-Ang2 Antibody or the antigen-bindingfragment thereof may not consist merely of at least one amino acidsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO: 4, and SEQ ID NO: 5.

In another embodiment, a method is provided for screening a candidateagent (anti-Ang2 antibody) for diagnosing, preventing, and/or treating adisease related to Ang2 overexpression, angiogenesis, increase ofvascular permeability, and/or a decrease of normal blood vesselformation using the above epitope, for example, a method for screeningan anti-Ang2 antibody. The screening method may comprise or consistessentially of:

(a) contacting a candidate compound to the three dimensional structureepitope of the aforementioned Angiopoietin-2; and

(b) measuring binding between the epitope and the candidate compound,for example, in the presence of a Tie2 receptor.

The screening method may further include step (c) of identifying bindingbetween Ang2 and Tie2 receptor. The step (c) is to identify whether thecandidate compound inhibits binding between Ang2 and Tie2 receptor andit may be performed before or after the step (b), or simultaneously withit. Through the step (c), an anti-Ang2 antibody which binds to Ang2 butdoes not inhibit binding between Ang2 and Tie2 receptor, i.e., having acharacteristic of inducing binding between Ang2 and Tie2 receptor can bescreened.

In the antibody screening method, when the binding between the epitopeand the candidate compound is detected, the candidate compound may bedetermined as a candidate agent (anti-Ang2 antibody). Therefore, theantibody screening method may further comprise, after step (b) or (c), astep of determining the candidate compound as a candidate agent(anti-Ang2 antibody) when the binding between the epitope and thecandidate compound is detected.

Furthermore, the screening method may further include step (d) ofidentifying the inhibition of Ang2 and/or the activation of Tie2receptor, and they can be included in any order. Through the step (d),an anti-Ang2 antibody which binds to Ang2 to inhibit the activitythereof but binds to Ang2 to induce binding between Ang2 and Tie2receptor and exhibits Tie2 receptor activation effects, like Ang1, canbe screened.

In the above screening method, when the epitope and the candidatecompound show binding affinity (kd) in the range of 10 nM or less, forexample, 0.1 pM to 10 nM, or pM to 10 nM, or 100 pM to 10 nM, or whenbinding between Ang2 and Tie2 receptor is identified (i.e., in case thatthe candidate compound does not inhibit binding between Ang2 and Tie2receptor) while the epitope and the candidate compound still show suchbinding affinity, the candidate compound can be determined to be acandidate for diagnosing, preventing, and/or treating a disease relatedto Ang2 overexpression, angiogenesis, increase of vascular permeability,and/or decrease of normal blood vessel formation, for example, ananti-Ang2 antibody candidate.

The epitope may be all or part (for example, at least one selected fromthe group consisting of the amino acid residue site exposed to theoutside of loop 1) of loop 1 (of SEQ ID NO: 11, a site from 417^(th)amino acid to 434^(th) amino acid) of human Ang2 (hAng-2; SEQ ID NO: 11;Accession # O15123) or an amino acid sequence site including 2 to 20, 2to 15, or 2 to 10 contiguous amino acids including at least one aminoacid residue exposed to the outside of loop 1 of SEQ ID NO: 11 and forexample, it may be at least one selected from the group consisting ofQ418, P419, a combination of Q418 and P419 positioned at loop 1 of SEQID NO: 11, or an amino acid sequence site including 2 to 20, 2 to 15, 2to 10, or 2 to 5 contiguous amino acids including the same.

The candidate compounds may be one or more selected from the groupconsisting of various artificially-synthesized or natural compounds,polypeptides, oligopeptides, peptide or protein scaffolds (for example,antibody, peptibody, nanobody, etc.), polynucleotides, oligonucleotides,antisense-RNA, shRNA (short hairpin RNA), siRNA (small interferenceRNA), aptamers, natural product extracts and so on.

The step of measuring the binding affinity between the epitope and thecandidate compound may be carried out using various methods known in theart. For example, the binding affinity may be measured using a Biacoremachine. In general, the range within which the binding affinity isconsidered as a therapeutic drug may be defined to have a bindingconstant KD value of not greater than 10 mM. For instance, in case thatthe binding affinity between the epitope of Angiopoietin-2 and aspecimen to be analyzed (for example, antibody) is 0.1 pM to 50 nM,particularly 0.5 pM to 35 nM, more particularly 1 pM to 10 nM whenmeasured using surface plasmon resonance methods such as a Biacoremachine, the specimen (for example, antibody) can be determined to be acandidate for diagnosing, preventing, and/or treating a disease relatedto Ang2 overexpression, angiogenesis, increase of vascular permeability,and/or decrease of normal blood vessel formation.

The identification of the presence of binding between Ang2 and Tie2 andthe identification of Ang2 inhibition and the activation of Tie2receptor will be described later.

In another embodiment, a polypeptide molecule is provided including theheavy chain complementarity determining region, the light chaincomplementarity determining region or the combination thereof; or theheavy chain variable region, the light chain variable region or thecombination thereof of the anti-Ang2 antibody as described above. Thepolypeptide molecule may function as a precursor or a component of anantagonist against Ang2 as well as for manufacturing an antibody or anantigen-binding fragment thereof. For example, the polypeptide moleculemay function as an Ang2 antigen binding site, and can be included as acomponent of a protein scaffold (e.g., peptibody, nanobody, etc.), abispecific antibody, and a multi-specific antibody having a similarstructure to an antibody.

The term “antagonist” as used herein is interpreted to encompass allmolecules that partially or entirely block, suppress or neutralize atleast one biological activity of its target (e.g., Ang2).

The term “peptibody (peptide+antibody)” used herein refers to a fusionprotein including a peptide and all or part of the constant region of anantibody such as an Fc portion wherein the peptide serves as an antigenbinding site (heavy chain and/or light chain CDR or variable regions)thereby to render a protein having similar framework and functions to anantibody

The term “nanobody” used herein is called a single-domain antibody,refers to an antibody fragment including a single variable domain of anantibody as a monomer form, and has characteristics of selectivelybinding to a specific antigen similarly to an antibody having an intactstructure. The molecular weight of the nanobody is generally about 12kDa to about 15 kDa, which is very little when compared to the normalmolecular weight (about 150 kDa or about 160 kDa) of an intact antibody(including two heavy chains and two light chains) and in some cases itis smaller than an Fab fragment or scFv fragment.

The term “bispecific antibody” or “multi-specific antibody” used hereinrefers to an antibody recognizing and/or binding to two (bispecificantibody) or more (multi-specific antibody) different antigens, orrecognizing and/or binding to different sites of the same antigen, andone antigen binding site of the bispecific antibody or multi-specificantibody may include the polypeptide described above.

In a specific embodiment, the polypeptide molecule may comprise orconsist essentially of:

at least one selected from the group consisting of a polypeptideincluding the amino acid sequence of SEQ ID NO: 1, a polypeptideincluding the amino acid sequence of SEQ ID NO: 2, and a polypeptideincluding the amino acid sequence of SEQ ID NO: 3;

at least one selected from the group consisting of a polypeptideincluding the amino acid sequence of SEQ ID NO: 4, a polypeptideincluding the amino acid sequence of SEQ ID NO: 5, and a polypeptideincluding the amino acid sequence of SEQ ID NO: 6; or

a combination thereof.

In a specific embodiment, the polypeptide molecule may comprise orconsist essentially of the amino acid sequence of SEQ ID NO: 8, theamino acid sequence of SEQ ID NO: 9, or a combination thereof.

In one embodiment, the polypeptide molecule may not consist merely of atleast one amino acid sequence selected from the group consisting of SEQID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5.

As described above, the above bispecific antibody or multi-specificantibody is referred to as an antibody including each antigen bindingsite to different two or more kinds of antigens and recognizing and/orbinding to the two or more kinds of antigens at the same time, whereinone of the antigen binding sites may include the aforementionedpolypeptide molecule. In particular, the polypeptide molecule serving asAng2 antigen binding site may form a dimer or multimer together with anantigen binding site to another antigen to constitute a bi-specificantibody or a multi-specific antibody. Accordingly, in one embodiment,there is provided a bi-specific antibody or a multi-specific antibodyincluding the polypeptide molecule as an Ang2 antigen binding site.

In another embodiment, there is provided a protein scaffold including atleast one (e.g., 1 to 5, or 2 to 4) peptide complex including one ormore of the aforementioned polypeptide molecules or a repeat where thepolypeptide molecules are repeatedly linked by a linker (hereafter,‘first peptide’), and a polypeptide having a structural function(hereafter, ‘second peptide’; e.g., a heavy chain or light chainconstant region of an antibody (IgG (IgG1, IgG2, IgG3, or IgG4), IgA,IgE, IgD, IgM, etc.), or an Fc fragment of an antibody) wherein said atleast one peptide complex is bound to at the second peptide (e.g., Fcfragment) to form a multimer structure.

Another embodiment provides a polynucleotide encoding the polypeptidemolecule, a recombinant vector comprising the polynucleotide, and arecombinant cell comprising (transfected with) the recombinant vector.

The antibody includes any animal-derived antibodies, chimericantibodies, humanized antibodies and human antibodies. An animal-derivedantibody which is produced by immunizing an animal with a desiredantigen may generally trigger an immune rejection response whenadministered to humans for treatment purpose, and a chimeric antibodyhas been developed to suppress such immune rejection response. Achimeric antibody is formed by replacing the constant region of ananimal-derived antibody, which is a cause of anti-isotype response, withthe constant region of a human antibody using genetic engineeringmethods. The chimeric antibody has considerably improved anti-isotyperesponse in comparison with animal-derived antibodies, butanimal-derived amino acids are still present in its variable regions andthus it still contains potential side effects resulting from ananti-idiotypic response. It is a humanized antibody that has been thusdeveloped to improve such side effects. This is manufactured by graftingCDR (complementarity determining regions) which, of the variable regionsof a chimeric antibody, has an important role in antigen binding into ahuman antibody framework.

An important consideration in CDR grafting technology for manufacturinga humanized antibody is to select an optimized human antibody which canreceive best the CDR of an animal-derived antibody and for this,utilization of antibody database, analysis of crystal structure,molecule modeling technology, etc. are employed. However, although theCDR of an animal-derived antibody is grafted into an optimized humanantibody framework, there are a considerable number of cases whereantigen binding affinity is not preserved because there are amino acidswhich affect antigen binding while being positioned at the framework ofthe animal-derived antibody. In this regard, it may be essential toapply an additional antibody engineering technology for restoringantigen binding affinity.

According to one embodiment, the antibody may be a mouse-derivedantibody, a mouse-human chimeric antibody, a humanized antibody, or ahuman antibody. The antibody or an antigen-binding fragment thereof maybe isolated from a living body or non-naturally occurring. The antibodyor an antigen-binding fragment thereof may be recombinant or synthetic.

Antibodies have been widely used for treating diseases. As antibodiesare very stable in vivo as well as in vitro and have a long half-life,they are favorable for mass expression and production. Also, since anantibody has intrinsically a dimer structure, it has a fairly highavidity.

An intact antibody has a structure with two full-length light chains andtwo full-length heavy chains, and each light chain is linked to eachheavy chain via a disulfide bond. The constant region of an antibody isdivided into a heavy chain constant region and a light chain constantregion, and the heavy chain constant region has gamma (γ), mu (μ), alpha(α), delta (δ) and epsilon (ε) types, and has gamma1 (γ1), gamma2 (γ2),gamma3 (γ3), gamma4 (γ4), alpha1 (α1) and alpha2 (α2) as its subclass.The light chain constant region has kappa (κ) and lambda (λ) types.

The term “heavy chain” is understood to include a full-length heavychain and fragments thereof, the full-length heavy chain including avariable region domain V_(H) including an amino acid sequence havingsufficient variable region sequences that contribute the specificity forantigen binding and three constant region domains C_(H1), C_(H2) andC_(H3) domains and a hinge. The term “light chain” is understood toinclude a full-length light chain and fragments thereof, the full-lengthlight chain including a variable region domain V_(L) including an aminoacid sequence having sufficient variable region sequences thatcontribute to the specificity for antigen binding and a constant regiondomain C_(L).

The term “CDR (complementarity determining region)” refers to an aminoacid sequence found in the hypervariable region of a heavy chain and alight chain of an immunoglobulin. The heavy and light chain may eachinclude three CDRs (CDRH1, CDRH2, CDRH3, and CDRL1, CDRL2, CDRL3). TheCDRs of an antibody can provide an essential contact residue for bindingto an antigen or an epitope. Throughout the specification, the terms“specifically binding” or “specifically recognizing” has the samemeaning as generally known to an ordinary person in the art, indicatingthat an antigen and an antibody specifically interact with each other tolead to an immunological response.

The antigen binding site of an antibody may be a fragment including atleast one complementarity determining region.

The term “antigen-binding fragment,” which is a fragment of the fullstructure of an immunoglobulin, refers to some of a polypeptideincluding a portion to which an antigen can bind. For example, it may bean scFv, an (scFv)₂, an scFv-Fc, an Fab, an Fab′ or an F(ab′)₂, but isnot limited thereto. Among the above antigen-binding fragments, an Fab,which is a structure having the light chain and heavy chain variableregions, the light chain constant region, and the heavy chain firstconstant region (C_(H1)), has one antigen binding site. An Fab′ differsfrom the Fab in that the Fab′ has a hinge region including at least onecysteine residue at the C-terminal of the heavy chain C_(H1) domain. AnF(ab′)₂ is produced when cysteine residues at the hinge region of Fab′are joined by a disulfide bond. An Fv is a minimal antibody fragment,having only heavy chain variable regions and light chain variableregions, and a recombinant technique for producing the Fv fragment iswell known in the art. In a two-chain Fv fragment, the heavy chainvariable domains are associated with the light chain variable domainsvia a non-covalent bond. A single-chain Fv fragment has a structure inwhich a heavy chain variable domain and a light chain variable domainare covalently joined to each other via a covalent bond or directly atthe C-terminus, so that it can form a dimer as in a two-chain Fvfragment. In this context, the heavy chain variable region and the lightchain variable region may be connected with each other through a linker,e.g., a peptide linker, or directly. The peptide linker may be composedof 1 to 100 amino acid residues, or 2 to 50 amino acid residues, with nolimitations imposed on the kind of the amino acid residues. For example,the peptide linker may include Gly, Asn and/or Ser, and may also includeneutral amino acids such as Thr and/or Ala. Amino acid sequencessuitable for use in the peptide linker may be those well known in theart. So long as it has no negative influence on the function of theantigen-binding fragment, the length of the peptide linker may beappropriately adjusted. For example, the peptide linker may be an aminosequence composed of 1 to 100, 2 to 50, or 5 to 25 amino acid residuesselected from among Gly, Asn, Ser, Thr, Ala, and a combination thereof.By way of example, the peptide linker may be (G4S)n (wherein nrepresents the repeating number of (G4S) and may be an integer of 1 to10, e.g., 2 to 5).

The antigen-binding fragment may be obtained using a protease (forexample, a whole antibody can be digested with papain to obtain Fabfragments, or can be digested with pepsin to obtain F(ab′)₂ fragments),or may be prepared by a genetic recombinant technique.

The term “hinge region” refers to a region included in the heavy chainsof an antibody, which is present between the CH1 and CH2 regions, andprovides flexibility to the antigen binding site in the antibody. Forexample, the hinge may be derived from a human antibody andparticularly, it may be derived from IgA, IgE, IgD, IgM, or IgG, forexample, IgG1, IgG2, IgG 3, or IgG4.

When an animal-derived antibody goes through a chimerization process, ananimal-derived IgG1 hinge is replaced with a human IgG1 hinge, but alength of the animal-derived IgG1 hinge is shorter than the human IgG1hinge, and disulfide bonds between two heavy chains are reduced from 3to 2. Thus, rigidity of the hinges may have different effects.Therefore, modification of a hinge region can increase an antigenbinding efficiency of a humanized antibody. Methods of deleting,inserting, or substituting an amino acid for modifying amino acidsequences of the hinge region are well known in the art.

Portions (e.g., constant regions) except the CDRs or variable regions ofthe anti-Ang2 antibody may be derived from a human antibody andparticularly, they may be derived from IgA, IgD, IgE, IgD, IgM, or IgG,for example, IgG1, IgG2, IgG 3, or IgG4.

The anti-Ang2 antibody may be a monoclonal antibody. The monoclonalantibody may be prepared by methods well known in the art. For example,it may be prepared using a phage display technique. Alternately, theAng2 antibody may be prepared into a mouse-derived monoclonal antibodyby methods set forth in the paper written by Schwaber, et al (Schwaber,J and Cohen, E. P., “Human×Mouse Somatic Cell Hybrid Clones SecretingImmunoglobulins of Both Parental Types,” Nature, 244 (1973), 444-447).

Meanwhile, individual monoclonal antibodies may be screened using atypical ELISA (Enzyme-Linked ImmunoSorbent Assay) format, based on thebinding potential with Ang2. Inhibitory activities can be verifiedthrough functional analysis such as competitive ELISA for verifying themolecular interaction of binding assemblies or functional analysis suchas a cell-based assay. Then, with regard to monoclonal antibody membersselected on the basis of their strong inhibitory activities, theiraffinities (Kd values) to Ang2 may be each verified.

The rest portions not including the antigen binding portions of thefinally selected antibodies may be prepared as not only humanimmunoglobulin antibodies but also humanized antibodies. Preparation ofhumanized antibodies is well known in the art (Almagro, J. C. andFransson, J., “Humanization of antibodies,” Frontiers in Bioscience, 13(2008), 1619-1633).

Another embodiment provides a hybridoma cell line which produces amonoclonal antibody of the anti-Ang2 antibody. The hybridoma cell linemay be a cell line having accession number (KCLRF-BP-00295).

As described above, the anti-Ang2 antibody or an antigen-bindingfragment thereof is characterized by binding to Ang2 while notinhibiting Ang2 from binding to Tie2 receptor and inducing bindingbetween Ang2 and Tie2. Also, a conjugate formed from binding of theanti-Ang2 antibody or an antigen-binding fragment thereof and Ang2 ischaracterized by binding to a Tie2 receptor (wherein an Ang2 portion ofthe conjugate participates in binding), just like Ang1, to activate theTie2 receptor.

Hence, another embodiment provides a conjugate of Ang2 and an anti-Ang2antibody in which the anti-Ang2 antibody or an antigen-binding fragmentthereof and Ang2 are bound to each other. Another embodiment provides acomposition for inducing binding of Ang2 with a Tie2 receptor, includingthe conjugate of Ang2 and an anti-Ang2 antibody as an active ingredient.Another embodiment provides a method for inducing binding of Ang2 with aTie2 receptor, including administering the conjugate of Ang2 and ananti-Ang2 antibody to a subject. The subject may be in need of bindingbetween Ang2 and Tie2 receptor. The method for inducing binding of Ang2with Tie2 receptor may further include a step of identifying a subjectwho is in need of binding between Ang2 and Tie2 receptor, prior to theadministration step. Still another embodiment provides a composition foractivating a Tie2 receptor including the conjugate of Ang2 and ananti-Ang2 antibody as an active ingredient. Still another embodimentprovides a method for activating a Tie2 receptor including administeringthe conjugate of Ang2 and an anti-Ang2 antibody to a subject. Thesubject may be in need of activating the Tie2 receptor. The Tie2receptor activation method may further include a step of identifying asubject who is in need of activating the Tie2 receptor, prior to theadministration step. The subject may be mammals including primates suchas humans and monkeys or rodents such as rats and mice, or cells,tissues, or body fluids (e.g., blood, serum, etc.) isolated therefrom orartificially cultured. Another embodiment provides a use of theconjugate of Ang2 and an anti-Ang2 antibody for activating a Tie2receptor.

As described above, since the anti-Ang2 antibody or an antigen-bindingfragment thereof has a function of inhibiting abnormal angiogenesis byinhibiting the functions of Ang2, it is applicable to prevent,alleviate, improve, and/or treat various diseases (e.g., cancer) relatedto abnormal angiogenesis (see Examples 13 and 14). Moreover, since theanti-Ang2 antibody or an antigen-binding fragment thereof does notinhibit binding between Ang2 and Tie2 (see Example 3 and FIG. 1), it canactivate a Tie2 signaling (see Example 6 and FIG. 3) by activating Tie2(see Example 6 and FIG. 3), and it accelerates the formation of vascularendothelium or lymphatic endothelium and increases mobility (see Example10) to suppress vascular permeability increase (see Example 11), wherebyit is applicable to prevent, alleviate, improve, and/or treat variousdiseases related to vascular permeability (for example, sepsis, eyedisorders, etc.). Also, as described above, since the anti-Ang2 antibodyor an antigen-binding fragment thereof accelerates the formation ofvascular endothelium or lymphatic endothelium to increase the formationof healthy blood vessels and normalize the blood vessels, it is alsoapplicable to prevent, alleviate, improve, and/or treat various diseasesor symptoms requiring the formation of healthy blood vessels such aswound healing or ischemic disorders, and it reduces cancer growth andmetastasis possibility by changing the abnormally formed cancer bloodvessels into structurally and functionally normal forms. Moreover, theanti-Ang2 antibody or an antigen-binding fragment thereof has an effectof suppressing inflammatory response (see Example 12), whereby it isapplicable to prevent, alleviate, improve, and/or treat variousinflammatory disorders. In addition, the anti-Ang2 antibody or anantigen-binding fragment thereof has the effect of normalization ofblood vessels in cancer cells (see Example 15), thereby increasing thetransporting efficiency of an anticancer agent through the normalizedblood vessels into Ang2 expressing cells. Therefore, whenco-administered with an anticancer agent to a subject, the anti-Ang2antibody or an antigen-binding fragment thereof can be used as anadjuvant for enhancing sensitiveness to the anticancer agent andefficacy of the anticancer agent in the subject (see Example 18).

Another embodiment provides a pharmaceutical composition for inhibitingangiogenesis including the anti-Ang2 antibody or an antigen-bindingfragment thereof as an active ingredient. Another embodiment provides amethod for inhibiting angiogenesis including administering the anti-Ang2antibody or an antigen-binding fragment thereof to a subject. Theanti-Ang2 antibody or an antigen-binding fragment thereof may beadministered in amount that is pharmaceutically effective, which amountmay be determined by the skilled medical practitioner or medicalresearcher. The subject may be in need of inhibiting angiogenesis. Theangiogenesis inhibition method may further include a step of identifyinga subject who is in need of the inhibition of angiogenesis, prior to theadministration step.

Another embodiment provides a pharmaceutical composition for reducingvascular permeability including the anti-Ang2 antibody or anantigen-binding fragment thereof as an active ingredient. Anotherembodiment provides a method for reducing vascular permeabilityincluding administering the anti-Ang2 antibody or an antigen-bindingfragment thereof to a subject. The anti-Ang2 antibody or anantigen-binding fragment thereof may be administered in amount that ispharmaceutically effective, which amount may be determined by theskilled medical practitioner or medical researcher. The subject may bein need of the reduction of vascular permeability. The vascularpermeability reduction method may further include a step of identifyinga subject who is in need of the reduction of vascular permeability,prior to the administration step.

Another embodiment provides a pharmaceutical composition for preventingand/or treating a disease related to Ang2 overexpression, angiogenesis,and/or vascular permeability increase including the anti-Ang2 antibodyor an antigen-binding fragment thereof as an active ingredient. Anotherembodiment provides a method for preventing and/or treating a diseaserelated to Ang2 overexpression, angiogenesis, and/or vascularpermeability increase including administering the anti-Ang2 antibody oran antigen-binding fragment thereof to a subject. The anti-Ang2 antibodyor an antigen-binding fragment thereof may be administered in amountthat is pharmaceutically effective, which amount may be determined bythe skilled medical practitioner or medical researcher. The subject maybe in need of preventing and/or treating the disease related to Ang2overexpression, angiogenesis, and/or vascular permeability increase. Theprevention and/or treatment method may further include a step ofidentifying a subject who is in need of preventing and/or treating adisease related to Ang2 overexpression, angiogenesis, and/or vascularpermeability increase, prior to the administration step.

Another embodiment provides a pharmaceutical composition for inducingnormal blood vessel formation including the anti-Ang2 antibody or anantigen-binding fragment thereof as an active ingredient. Anotherembodiment provides a method of inducing normal blood vessel formation,including administering the anti-Ang2 antibody or an antigen-bindingfragment thereof to a subject. The anti-Ang2 antibody or anantigen-binding fragment thereof may be administered in amount that ispharmaceutically effective, which amount may be determined by theskilled medical practitioner or medical researcher. The subject may bein need of inducing normal blood vessel formation. The method ofinducing normal blood vessel formation may further include a step ofidentifying a subject who is in need of inducing normal blood vesselformation, prior to the administration step.

Another embodiment provides a pharmaceutical composition for preventingand/or treating a disease related to normal blood vessel formationdecrease including the anti-Ang2 antibody or an antigen-binding fragmentthereof as an active ingredient. Another embodiment provides a methodfor preventing and/or treating a disease related to normal blood vesselformation decrease including administering the anti-Ang2 antibody or anantigen-binding fragment thereof to a subject. The anti-Ang2 antibody oran antigen-binding fragment thereof may be administered in amount thatis pharmaceutically effective, which amount may be determined by theskilled medical practitioner or medical researcher. The subject may bein need of preventing and/or treating the disease related to normalblood vessel formation decrease. The prevention and/or treatment methodmay further include a step of identifying a subject who is in need ofpreventing and/or treating a disease related to normal blood vesselformation decrease, prior to the administration step.

Another embodiment provides a pharmaceutical composition for preventingand/or treating a cancer including the anti-Ang2 antibody or anantigen-binding fragment thereof as an active ingredient. Anotherembodiment provides a method for preventing and/or treating a cancerincluding administering the anti-Ang2 antibody or an antigen-bindingfragment thereof to a subject. The anti-Ang2 antibody or anantigen-binding fragment thereof may be administered in amount that ispharmaceutically effective, which amount may be determined by theskilled medical practitioner or medical researcher. The subject may bein need of preventing and/or treating a cancer. The prevention and/ortreatment method may further include a step of identifying a subject whois in need of preventing and/or treating a disease related to normalblood vessel formation decrease, prior to the administration step.

Another embodiment provides a pharmaceutical composition for tissueregeneration and/or wound healing including the anti-Ang2 antibody or anantigen-binding fragment thereof as an active ingredient. Anotherembodiment provides a method for tissue regeneration and/or woundhealing including administering the anti-Ang2 antibody or anantigen-binding fragment thereof to a subject. The anti-Ang2 antibody oran antigen-binding fragment thereof may be administered in amount thatis pharmaceutically effective, which amount may be determined by theskilled medical practitioner or medical researcher. The subject may bein need of tissue regeneration and/or wound healing. The method mayfurther include a step of identifying a subject who is in need of tissueregeneration and/or wound healing, prior to the administration step. Asubject to whom the active ingredient is administered may be a subjectwho has a skin tissue or organ tissue damage or has received a skintransplant.

Another embodiment provides a pharmaceutical composition for inhibitingAng2 and/or activating a Tie2 receptor including the anti-Ang2 antibodyor an antigen-binding fragment thereof as an active ingredient. Anotherembodiment provides a method for inhibiting Ang2 and/or activating aTie2 receptor including administering the anti-Ang2 antibody or anantigen-binding fragment thereof to a subject. The anti-Ang2 antibody oran antigen-binding fragment thereof may be administered in amount thatis pharmaceutically effective, which amount may be determined by theskilled medical practitioner or medical researcher. The subject may bein need of Ang2 inhibition and/or Tie2 receptor activation. The Ang2inhibition and/or Tie2 receptor activation method may further include astep of identifying a subject who is in need of Ang2 inhibition and/orTie2 receptor activation, prior to the administration step. Theanti-Ang2 antibody or an antigen-binding fragment thereof may be in aform of being bound to an antigen Ang2.

Since the function of the anti-Ang2 antibody or an antigen-bindingfragment thereof, which is an active ingredient of the abovepharmaceutical compositions, is activated by the binding with Ang2, thepharmaceutical compositions may further include Ang2 to enhance thefunction of the antibody or the antigen-binding fragment thereof. Theabove methods may further include a step of administering Ang2 to asubject. The Ang2 may be administered in amount that is pharmaceuticallyeffective, which amount may be determined by the skilled medicalpractitioner or medical researcher. The Ang2 may be administeredtogether with the anti-Ang2 antibody or an antigen-binding fragmentthereof simultaneously or sequentially in any order.

Another embodiment provides a pharmaceutical composition (an adjuvant)for enhancing the efficacy of an anticancer agent including theanti-Ang2 antibody or an antigen-binding fragment thereof. Anotherembodiment provides a method of enhancing the efficacy of an anticanceragent. The method includes administering the anti-Ang2 antibody or anantigen-binding fragment thereof to a subject in need of administrationof the anticancer agent. The anti-Ang2 antibody or an antigen-bindingfragment thereof may be administered in amount that is pharmaceuticallyeffective, which amount may be determined by the skilled medicalpractitioner or medical researcher. The method may further include,prior to the administration step, a step of identifying a subject inneed of administration of the anticancer agent. The enhancement of ananticancer agent may refer to making the anticancer agent exhibit moreexcellent anticancer effect at relatively low dosage, which is due tovascular normalization thereby increasing transporting efficiency of theanticancer agent into cancer tissues and increasing sensitiveness to theanticancer agent. The adjuvant may refer to a supplementarypharmaceutical composition used for enhancing the efficacy of ananticancer agent.

An embodiment provides a pharmaceutical composition for combinationtherapy for preventing and/or treating of a cancer, including ananticancer agent and the anti-Ang2 antibody or an antigen-bindingfragment thereof as active ingredients.

The pharmaceutical composition for combination therapy may be a mixedformulation (e.g., a single composition comprising two or more activeingredients) of the anticancer agent and the anti-Ang2 antibody or anantigen-binding fragment thereof. The anticancer agent and the anti-Ang2antibody or an antigen-binding fragment thereof can be present in anyamount that is pharmaceutically effective when used together. Thecomposition thus formulated can be used for simultaneous administrationof the two active ingredients.

Alternatively, the anticancer agent and the anti-Ang2 antibody or anantigen-binding fragment thereof can each be formulated in a separatecomposition, and the two active ingredients can be separatelyadministered simultaneously or sequentially. For instance, a firstpharmaceutical composition including a pharmaceutically effective amountof the anticancer agent as an active ingredient and a secondpharmaceutical composition including a pharmaceutically effective amountthe anti-Ang2 antibody or an antigen-binding fragment thereof as anactive ingredient can be administered simultaneously or sequentially. Inthe case of the sequential administration, any order of administrationmay be used.

Another embodiment provides a kit useful for preventing and/or treatinga cancer, including a first pharmaceutical composition including ananticancer agent as an active ingredient, a second pharmaceuticalcomposition including the anti-Ang2 antibody or an antigen-bindingfragment thereof as an active ingredient, and a package container. Theanticancer agent and the anti-Ang2 antibody or an antigen-bindingfragment thereof may be used in amounts that are pharmaceuticallyeffective when combined, which amount may be determined by the skilledmedical practitioner or medical researcher. The package container can beany container that holds or otherwise links the two compositions inindividual containers together in a single unit (e.g., a box that holdsboth containers, or plastic wrap that binds both containers together),or the package container may be a single, divided container having atleast two chambers that each hold one of the two compositions.

A method of combination therapy for preventing and/or treating a canceralso is provided. The method includes co-administering an anticanceragent and the anti-Ang2 antibody or an antigen-binding fragment thereofto a subject in need of the prevention and/or treatment of cancer. Theanticancer agent and the anti-Ang2 antibody or an antigen-bindingfragment thereof may be administered in amounts that arepharmaceutically effective when combined, which amount may be determinedby the skilled medical practitioner or medical researcher. The methodmay further include, prior to the co-administration step, a step ofidentifying a subject in need of the prevention and/or treatment ofcancer. The step of identifying may be conducted by any manners and/ormethods known to relevant field for identifying whether or not a subjectneeds the prevention and/or treatment of cancer. For example, the stepof identifying may include diagnosing a subject to have a cancer, oridentifying a subject who is diagnosed as a cancer subject. In thiscase, the anti-Ang2 antibody or an antigen-binding fragment thereof maybe in a form combined form with Ang2.

The co-administration may be conducted by administering the anticanceragent and the anti-Ang2 antibody or an antigen-binding fragment thereofsimultaneously or sequentially in any order. In one embodiment, theco-administration may be conducted by administering a mixed formulationof the anticancer agent and the anti-Ang2 antibody or an antigen-bindingfragment thereof. In another embodiment, the co-administration may beconducted by a first step of administering the anticancer agent, and asecond step of administering the anti-Ang2 antibody or anantigen-binding fragment thereof, wherein the first and the secondadministration steps may be conducted simultaneously or sequentially. Incase of the sequential administration, the first step and the secondstep may be performed in any order. The anticancer agent and theanti-Ang2 antibody or an antigen-binding fragment thereof may beadministered in amounts that are pharmaceutically effective whencombined, which amount may be determined by the skilled medicalpractitioner or medical researcher.

The anticancer agent may be at least one selected from all chemicaldrugs, antibodies, genes (e.g., antisense oligonucleotide, siRNA, shRNA,microRNA, etc.), aptamer, cells for therapeutic use, agents forradiotherapy, and the like, for the use of growth inhibition, apoptosispromotion and metastasis inhibition of cancer cells or cancer tissues.For example, the anticancer agent may be at least one selected from thegroup consisting of, but not limited thereto:

1) alkylating agents including i) platinum-based compounds includingcisplatin, carboplatin, oxaliplatin (oxliplatin), and the like, ii)nitrogen mustard-based compounds including mechlorethamine (nitrogenmustard), cyclophosphamide, ifosfamide, melphalan, chlorambucil, and thelike, iii) ethylenimine- and methylmelamine-based drugs includingthiotepa, altretamine, and the like, iv) methylhydrazine derivativesincluding procarbazine, and the like, v) alkyl sulfonate-based drugincluding busulfan, and the like, vi) nitrosourea-based drugs includingcarmustine, lomustine, and the like, and vii) triazine-based drugsincluding dacarbazine, and the like;

2) antimetabolites including i) pyrimidine derivatives includingfluorouracil (5-FU), capecitabine, cytarabine, gemcitabine, and thelike, ii) folic acid derivatives including methotrexate(MTX), and thelike, and iii) purine derivatives including mercaptopurine (6-MP), andthe like;

3) natural materials including i) vinca alkaloid including vinblastine,vincristine, vinorelvine, and the like, ii) taxane including paclitaxel,docetaxel, and the like, iii) epipodophyllotoxin including etoposide andthe like, and iii) camptothecin including topotecan, irinotecan, and thelike;

4) antibiotic materials including dactinomycin, doxorubicin,daunorubicin, mitomycin, bleomycin, and the like;

5) Targeted drugs including i) tyrosine kinase inhibitors includingimatinib, trastuzumab, cetuximab, gefitinib, erlotinib, and the like,and ii) angiogenesis inhibitors including bevacizumab, sunitinib,sorafenib, cabozantinib, pazopanib, regorafenib, vandetanib,ziv-afilibercept, and the like; and

6) prednisone, 6-thioguanine (6-TG), and the like.

The cancer may be a solid cancer or blood cancer. For instance, thecancer may be, not limited to, at least one selected from the groupconsisting of squamous cell carcinoma, small-cell lung cancer,non-small-cell lung cancer, adenocarcinoma of the lung, squamous cellcarcinoma of the lung, peritoneal carcinoma, skin cancer, melanoma inthe skin or eyeball, rectal cancer, cancer near the anus, esophaguscancer, small intestinal tumor, endocrine gland cancer, parathyroidcancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic oracute leukemia, lymphocytic lymphoma, hepatoma, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatocellular adenoma, breast cancer, coloncancer, large intestine cancer, endometrial carcinoma or uterinecarcinoma, salivary gland tumor, kidney cancer, prostate cancer, vulvarcancer, thyroid cancer, head and neck cancers, brain cancer,osteosarcoma and so on. The cancer may include a metastatic cancer aswell as a primary cancer.

Since the anti-Ang2 antibody or an antigen-binding fragment thereof isactivated by binding with Ang2, the composition may further include Ang2for enhancing the function of the antibody or an antigen-bindingfragment thereof. In addition, the method may further include a step ofadministering a pharmaceutically effective of Ang2 to a subject.

Another embodiment provides a composition for diagnosing a diseaserelated to angiogenesis and/or increase of vascular permeability and/ordecrease of normal blood vessel formation decrease, including theanti-Ang2 antibody or an antigen-binding fragment thereof. Anotherembodiment provides a method of diagnosing a disease related toangiogenesis and/or increase of vascular permeability and/or decrease ofnormal blood vessel formation decrease, including treating a biologicalsample derived from a subject with the anti-Ang2 antibody or anantigen-binding fragment thereof, and measuring a level of anantigen-antibody reaction. In this method, when the level of theantigen-antibody reaction in the biological sample is higher than thatof a normal sample, the subject from which the biological sample isderived may be determined as having diagnosing a disease related toangiogenesis and/or increase of vascular permeability and/or decrease ofnormal blood vessel formation decrease and/or vascular leakage and/orvascular inflammation. Therefore, the method may further includetreating a normal sample with the anti-Ang2 antibody or anantigen-binding fragment thereof, and measuring a level of anantigen-antibody reaction. Another embodiment provides a use of theanti-Ang2 antibody or an antigen-binding fragment thereof for diagnosinga disease related to angiogenesis and/or increase of vascularpermeability and/or decrease of normal blood vessel formation decrease.

The biological sample may be at least one selected from the groupconsisting of a cell, a tissue, fluid (e.g., blood, serum, and the like)and the like, derived from a subject to be diagnosed. The biologicalsample may be separated from a living body. The normal sample may be atleast one selected from the group consisting of a cell, a tissue, fluid(e.g., blood, serum, and the like) and the like, derived from a subjecthaving no disease related to angiogenesis, and/or vascular permeabilityincrease and/or normal blood vessel formation decrease. The normalsample may be separated from a living body. The subject may be selectedfrom mammal including primates such as a human, a monkey, and the like,and rodents such as a mouse, a rat, and the like.

The pharmaceutical compositions may further include a pharmaceuticallyacceptable carrier, and the carrier may be those commonly used in theformulation of drugs, which may be one or more selected from the groupconsisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch,gum acacia, calcium phosphate, alginates, gelatin, calcium silicate,micro-crystalline cellulose, polyvinylpyrrolidone, cellulose, water,syrup, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate,talc, magnesium stearate, and mineral oil, but is not limited thereto.The pharmaceutical compositions may further include one or more selectedfrom the group consisting of a diluent, an excipient, a lubricant, awetting agent, a sweetener, a flavor enhancer, an emulsifying agent, asuspension agent, and a preservative.

Pharmaceutically effective amounts of the pharmaceutical compositions,or the antibody or the antigen-binding fragment thereof may beadministered orally or parenterally. The parenteral administration mayinclude intravenous injection, subcutaneous injection, muscularinjection, intraperitoneal injection, endothelial administration, localadministration, intranasal administration, intrapulmonaryadministration, and rectal administration. Since oral administrationleads to digestion of proteins or peptides, an active ingredient in thecompositions for oral administration must be coated or formulated toprevent digestion in stomach. In addition, the composition may beadministered using an optional device that enables an active substanceto be delivered to target cells.

The content of the anti-Ang2 antibody or an antigen-binding fragmentthereof in the pharmaceutical compositions may be prescribed in avariety of ways, depending on factors such as formulation methods,administration methods, age of subjects, body weight, gender, pathologicconditions, diets, administration time, administration interval,administration route, excretion speed, and reaction sensitivity. Forexample, a daily dosage of the anti-Ang2 antibody or an antigen-bindingfragment thereof may be within the range of 0.001 to 1000 mg/kg,particularly 0.01 to 100 mg/kg, and more particularly 0.1 to 50 mg/kg,but is not limited thereto. The daily dosage may be formulated into asingle formulation in a unit dosage form or formulated in suitablydivided dosage forms, or it may be manufactured to be contained in amultiple dosage container. The term “pharmaceutically effective amount”as used herein refers to a content or dose of an active ingredientcapable of showing desirable pharmacological effects and it may bedetermined in a variety of ways, depending on factors such asformulation methods, administration methods, age of subjects, bodyweight, gender, pathologic conditions, diets, administration time,administration interval, administration route, excretion speed, andreaction sensitivity.

The pharmaceutical compositions may be formulated into a form of asolution in oil or an aqueous medium, a suspension, syrup, anemulsifying solution, an extract, powder, granules, a tablet, or acapsule, and may further include a dispersing or a stabilizing agent forthe formulation.

In particular, the pharmaceutical compositions including the anti-Ang2antibody or an antigen-binding fragment thereof may be formulated intoan immunoliposome since it contains an antibody or an antigen-bindingfragment. A liposome containing an antibody may be prepared using anymethods widely known in the art. The immunoliposome may be a lipidcomposition including phosphatidylcholine, cholesterol, andpolyethyleneglycol-derivatized phosphatidylethanolamine, and may beprepared by a reverse phase evaporation method. For example, Fab′fragments of an antibody may be conjugated to the liposome through adisulfide-exchange reaction.

Meanwhile, as the anti-Ang2 antibody or an antigen-binding fragmentthereof specifically binds to Ang2, this can be used to detect Ang2, andthe presence of the overexpression of Ang2 can be verified through it.Accordingly, another embodiment provides a composition for detectingAng2 and a composition for diagnosing a disease related to Ang2overexpression including the anti-Ang2 antibody or an antigen-bindingfragment thereof.

In another embodiment, a method is provided for detecting Ang2 includingtreating a living specimen obtained (or isolated) from a subject withthe anti-Ang2 antibody or an antigen-binding fragment thereof; andidentifying the presence of an antigen-antibody reaction. Also, there isprovided a method of providing information for diagnosis of a diseaserelated to Ang2 overexpression, including treating a living specimenobtained from a subject with the anti-Ang2 antibody or anantigen-binding fragment thereof; and identifying the presence of anantigen-antibody reaction. The method of providing information for thediagnosis may determine a subject to have Ang2 overexpression symptoms,or have Ang2 overexpression related diseases when an antigen-antibodyreaction is detected in the step of identifying the presence of anantigen-antibody reaction. The living specimen may be selected from thegroup consisting of cells, tissues and body fluids obtained (isolated)from a subject.

The step of identifying the presence of the antigen-antibody reactionmay be performed using various methods known in the art. For example, itmay be measured through an ordinary enzyme reaction, fluorescence,luminescence, and/or radioactivity detection and particularly, it may bemeasured by a method selected from the group consisting ofimmunochromatography, immunohistochemistry, enzyme linked immunosorbentassay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA),fluorescence immunoassay (FIA), luminescence immunoassay (LIA), westernblotting, etc., but is not limited thereto.

The subjects which the pharmaceutical composition or the antibody or theantigen-binding fragment thereof is administered to or is aimed todiagnose may be mammals including primates such as humans and monkeys,or rodents such as rats and mice, or cells, tissues and body fluidsisolated therefrom or artificially cultured.

The diseases related to angiogenesis and/or vascular permeabilityincrease and/or Ang2 overexpression may be cancer; cancer metastasis;ocular blood vessel disorders such as retinopathy of prematurity,macular degeneration (e.g., age-related macular degeneration), diabeticretinopathy, neovascular glaucoma, etc.; inflammatory disorders such aspsoriasis, asthma, rheumatoid arthritis, pneumonia, chronicinflammation, etc.; infectious disorders (infection); cardiovasculardisorders such as hypertension, arteriosclerosis, etc.; renal disease;sepsis; asthma; edema; hereditary hemorrhagic telangiectasia (HHT), etc.The cancer may be those overexpressing Ang2, it may be a solid cancer ora blood cancer, and it may be, but not limited to, selected from thegroup consisting of squamous cell carcinoma, small-cell lung cancer,non-small-cell lung cancer, adenocarcinoma of the lung, squamous cellcarcinoma of the lung, peritoneal carcinoma, skin cancer, melanoma inthe skin or eyeball, rectal cancer, cancer near the anus, esophaguscancer, small intestinal tumor, endocrine gland cancer, parathyroidcancer, adrenal cancer, soft-tissue sarcoma, urethral cancer, chronic oracute leukemia, lymphocytic lymphoma, hepatocellular cancer, gastriccancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, liver cancer, bladder cancer, hepatoma, breast cancer, coloncancer, large intestine cancer, endometrial carcinoma or uterinecarcinoma, salivary gland tumor, kidney cancer, prostate cancer, vulvarcancer, thyroid cancer, head or neck cancer, brain cancer, osteosarcoma,etc.

The diseases related to normal blood vessel formation decrease arediseases that require the induction of normal blood vessel formation andmay be selected from the group consisting of ischemic disorders such asmyocardial infarction, angina, cerebral infarction, stroke (ischemicstroke), etc., Buerger' disease (thromboangiitis obliterans), avascularnecrosis, foot ulcer (e.g., diabetic foot ulcer), erectile dysfunctionand so on.

Another embodiment provides a complex in which the anti-Ang2 antibody oran antigen-binding fragment thereof, Ang2, and Tie2 receptor are bound.The complex may be present inside the body or present in a cell isolatedfrom the body. Also, the antibody in the complex may form a dimer withanother antibody in an adjacent complex, thereby clustering two or morecomplexes, to form a cluster including two or more complexes (seeExample 8). Such action is related to the Tie2 receptor activationfunction of the anti-Ang2 antibody. The complex can be used to monitorthe action of the anti-Ang2 antibody, i.e., the presence of Ang2inhibition and/or Tie2 receptor activation, or simply used to inhibitAng2 and/or to activate Tie2 receptor.

Another embodiment provides a method for screening a candidate drug forpreventing and/or treating a disease related to angiogenesis and/orincrease of vascular permeability by identifying the complex formation,and/or the presence of Ang2 inhibition and/or Tie2 receptor activation.

In one embodiment, the screening method may comprise or consistessentially of

contacting a candidate compound to a specimen containing Ang2 and a Tie2receptor; and

identifying the formation of a complex in which the candidate compound,Ang2, and the Tie2 receptor are bound.

The step of identifying the formation of a complex in which thecandidate compound, Ang2, and the Tie2 receptor are bound may beperformed by identifying the presence of a complex in which thecandidate compound, Ang2, and the Tie2 receptor are bound, oridentifying the presence of binding between the candidate compound andAng2 and the presence of binding between Ang2 and the Tie2 receptor. Thescreening method may further include a step of identifying Ang2inhibition and/or Tie2 receptor activation, before/after the step ofidentifying the formation of a complex in which the candidate compound,Ang2, and the Tie2 receptor are bound, or simultaneously therewith.

In the above screening method, when the formation of a complex in whichthe candidate compound, Ang2, and the Tie2 receptor are bound isidentified, in other words, in case that the presence of a complex inwhich the candidate compound, Ang2, and the Tie2 receptor are bound isidentified, or in case that binding between the candidate compound andAng2 and binding between Ang2 and the Tie2 receptor are identified, thecandidate compound can be determined to be a candidate for preventingand/or treating a disease related to Ang2 overexpression, angiogenesis,increase of vascular permeability, and/or decrease of normal bloodvessel formation.

In another embodiment, the screening method may comprise or consistessentially of

contacting a candidate compound to a specimen containing Ang2 and a Tie2receptor; and

identifying Ang2 inhibition and/or Tie2 receptor activation (i.e., Ang2inhibition, Tie2 receptor activation, or a combination thereof). In thisscreening method, when Ang2 inhibition and/or Tie2 receptor activationis identified, the candidate compound can be determined to be acandidate for preventing and/or treating a disease related to Ang2overexpression, angiogenesis, increase of vascular permeability, and/ordecrease of normal blood vessel formation. The inhibition (degradation)of Ang2 may be identified by measuring the levels of Ang2 before/afterthe same specimen is treated with Ang2, or measuring the levels of Ang2after the same specimen is divided into a treatment group and anon-treatment group, and then comparing the levels of Ang2 before/afterthe treatment of the candidate compound or in the treatment group andthe non-treatment group. When the level of Ang2 after the treatment ofthe candidate compound or in the treatment group is decreased, incomparison with the pre-treatment of the candidate compound or thenon-treatment group, Ang2 may be determined to be inhibited. Also, theactivation of Tie2 receptor may be identified by measuring thephosphorylation degrees of the Tie2 receptor and/or the phosphorylationdegree of at least one protein involved in the downstream signaling ofthe Tie2 receptor (for example, Akt, eNOS, 42/44, etc.) before/after thesame specimen is treated with the candidate compound, or measuring themafter the same specimen is divided into a treatment group and anon-treatment group, and then comparing the phosphorylation degrees ofthe proteins before/after the treatment of the candidate compound or inthe treatment group and the non-treatment group. When thephosphorylation degree after the treatment of the candidate compound orin the treatment group is increased, in comparison with thepre-treatment of the candidate compound or the non-treatment group, theTie2 receptor may be determined to be activated. Also, the activation ofthe Tie2 receptor may be determined by identifying the presence of theintracellular internalization of the Tie2 receptor.

In another embodiment, the screening method may comprise or consistessentially of

contacting a candidate compound to a specimen containing Ang2 and a Tie2receptor; and

identifying the formation of a complex in which the candidate compound,Ang2, and the Tie2 receptor are bound, and Ang2 inhibition and/or Tie2receptor activation.

The identification of the complex formation, the measurement of Ang2level, the measurement of Tie2 phosphorylation degree, the measurementof the phosphorylation degree of the proteins involved in downstreamsignaling. and/or the identification of the intracellularinternalization of Tie2 receptor may be performed using various methodsknown in the art and for example, they may be measured through anordinary enzyme reaction, fluorescence, luminescence, and/orradioactivity detection and particularly, they may be measured by amethod selected from the group consisting of immunochromatography,immunohistochemistry, enzyme linked immunosorbent assay (ELISA),radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescenceimmunoassay (FIA), luminescence immunoassay (LIA), western blotting,etc., but is not limited thereto.

The candidate compounds may be one or more selected from the groupconsisting of various artificially-synthesized or natural compounds,polypeptides, oligopeptides, peptide or protein scaffolds (for example,antibody, peptibody, nanobody, etc.), polynucleotides, oligonucleotides,antisense-RNA, shRNA (short hairpin RNA), siRNA (small interferenceRNA), aptamers, natural product extracts and so on.

The specimen containing Ang2 and Tie2 receptor may be cells or tissuesisolated from a living body or artificially cultured, and they mayintrinsically contain (express) Ang2 and the Tie2 receptor, may betreated with Ang2 and/or the Tie2 receptor, or may be manipulated toexpress Ang2 and/or the Tie2 receptor.

Previously, there were attempts to inhibit angiogenesis by Ang2 byinhibiting binding between Ang2 and its receptor Tie2, but an antibodywhich specifically binds to Ang2 to induce the intracellularinternalization and degradation of Ang2 and at the same time, maintainsa binding ability with Ang2 and Tie2 receptor to form anantibody-Ang2-Tie2 receptor complex, thereby activating the Tie2receptor has not been known so far. Under such circumstances, theinvention proposes a novel method capable of inhibiting angiogenesis byAng2 and reducing vascular permeability by suggesting an antibody whichinhibits Ang2 and at the same time activates the Tie2 receptor toaccelerate its downstream signaling. Also, the antibody proposed in theinvention is anticipated to be applicable to diagnose and treat abnormalblood vessel formation-related disorders other than cancer and/ordisorders caused by vascular permeability increase. The antibody can beutilized for combination therapy with chemical medicines and otheranticancer drugs, and is expected to be employed for antibody fragments,bi- or multi-specific antibodies, protein scaffolds, etc. using Ang2specific recognition activity.

Hereafter, the present invention will be described in detail byexamples. They are intended merely to illustrate the invention and arenot construed to restrict the invention. It will be obvious to thoseskilled in the pertinent art that the following examples may be modifiedwithin the scope of not deviating from the essential gist of theinvention.

Example 1 Preparation of Anti-Ang2 Antibody

A human Ang2 protein (R&D systems; 623-AN-025/CF) was administered to5-week-old BALB/c mice along with an adjuvant to induce an immuneresponse and then, hybridomas that produce an individual anti-Ang2antibody were prepared according to the known methods described in thepaper written by Schwaber, et al (Schwaber, J and Cohen, E. P.,“Human×Mouse Somatic Cell Hybrid Clones Secreting Immunoglobulins ofBoth Parental Types,” Nature, 244 (1973), 444-447).

More specifically, to obtain immunized mice necessary for developinghybridoma cell lines, 100 ug (microgram) of human Ang2 protein (R&DSystems) mixed with the same amount of a complete Freund's adjuvant wasadministered via an intraperitoneal injection to each of five4˜6-week-old BALB/c mice (Japan SLC, Inc.). After two weeks, the antigen(half the previously injected amount) mixed with an incomplete Freund'sadjuvant using the same method as described above was administered toeach mouse via an intraperitoneal injection. After one additional week,a final boosting was performed and three days later, blood was collectedfrom the tail of each mouse to obtain serum, which was then diluted at1/1000 with PBS and subjected to an ELISA to verify that the titer of anantibody recognizing Ang2 was increased. From the results, mice in whicha sufficient amount of the antibody was obtained were selected, and acell fusion process was performed on the selected mice.

Three days before the cell fusion experiment, a mixture of 50 ug of PBSand 100 ug of human Ang2 protein (R&D systems) was administered via anintraperitoneal injection to BALB/c mice (Japan SLC, Inc.), and aftereach immunized mouse was anesthetized, its spleen located on the leftside of the body was extracted. The extracted spleen was ground with amesh to isolate cells, which were mixed with a culture medium (DMEM,Hyclon) to prepare a spleen cell suspension. The suspension wascentrifuged to collect a cell layer. The obtained 1×10⁸ spleen cellswere mixed with 1×10⁷ myeloma cells (Sp2/0), and the mixture wascentrifuged to precipitate the cells. The centrifuged precipitate wasslowly dispersed, treated with 1 ml of 45% polyethylene glycol (PEG1500) contained in a culture medium (DMEM), and maintained at 37° C. forone minute before adding 1 ml of a culture medium (DMEM). Subsequently,10 ml of the culture medium (DMEM) was added for 1 minute to theresultant, which was incubated in a water bath at 37° C. for 5 minutesand then re-centrifuged after the total volume was adjusted to 50 ml.The resulting cell precipitate was re-suspended in an isolation medium(HAT medium) at a concentration of 1˜2×10⁵/ml, and the resultantsuspension was distributed at 0.1 ml to the each well of a 96-wellplate, which was then incubated in a carbon dioxide incubator at 37° C.to prepare the hybridoma cell groups.

Example 2 Manufacture of Anti-Ang2 Antibody 2.1. Selection of Anti-Ang2Antibody Producing Clone and Purification of Antibody

The above obtained individual antibody producing hybridomas werescreened using a typical ELISA format to select hybridomas which produce95 anti-Ang2 monoclonal antibodies among the hybridomas differentiatedfrom their mother hybridomas, based on their binding potential withAng2.

More specifically, to select the hybridoma cells that specifically reactonly to Ang2 protein among the hybridoma cell groups prepared in Example1 above, an ELISA assay method using a human Ang2 protein as an antigenwas used for screening.

Human Ang2 protein was added at 100 ng per well to a microtiter plate tobe adhered to the surface of the plate, and unreacted antigens wereremoved by washing. 50 microliters of the hybridoma cell cultureobtained in Example 1 above was added to each well to react for 1 hourand then, the wells were sufficiently washed with phosphate bufferedsaline-TWEEN 20 (PBST) solution to remove unreacted culture solution.Goat anti-mouse IgG-horseradish peroxidase (goat anti-mouse IgG-HRP) wasadded thereto, a reaction was allowed to occur at a room temperature for1 hour and then, washing was sufficiently performed with the TBSTsolution. Subsequently, substrate solution (OPD) of peroxidase was addedto each well to react, and the reaction degree was measured by theabsorption at 450 nm using an ELISA reader to repeatedly selecthybridoma cell lines that secret antibodies having specifically highbinding affinity only to human Ang2 protein. A limiting dilution wasperformed on the hybridoma cell lines obtained through repetitiveselection to obtain final 58 clones of hybridoma cell lines producingmonoclonal antibodies. The thus prepared hybridomas were deposited inthe Korean Cell Line Bank located at Yongon-dong, Chongno-gu, Seoul,South Korea, as of Apr. 23, 2013 and received accession numberKCLRF-BP-00295.

Each hybridoma obtained above was cultured in DMEM (Dulbeco's ModifiedEagle's Medium) and then, the culture solutions were collected andsubjected to Protein G-affinity chromatography method to purifyanti-Ang2 monoclonal antibodies produced from each hybridoma.

First, the hybridoma cells cultured in 50 ml of culture medium (DMEM)containing 10% (v/v) FBS were centrifuged to obtain a cell precipitate,which was washed at least twice with 20 ml of PBS to remove the FBS. Thecell precipitate was re-suspended in 50 ml of the culture medium (DMEM)and then incubated in a carbon dioxide incubator at 37° C. for 3 days.Subsequently, the cell culture was centrifuged to remove theantibody-producing cells, and the culture medium including the secretedantibodies was isolated and then, stored at 4° C. or used directly.Antibodies were purified from 50 to 300 ml of the culture medium usingan AKTA purification device (GE Healthcare) equipped with an affinitycolumn (protein G agarose column; Pharmacia, USA). The purifiedantibodies were stored for subsequent use after replacing thesupernatant with PBS using a filter for protein aggregation (Amicon).One of the antibodies obtained from each hybridoma above was named 10D6.

The binding affinity of the above antibody to human Ang2 protein wasmeasured by an SPR method using a BIAcore T100 (GE Healthcare). The SPRmethod uses refractive index change of light which passes a sensor chipaccording to the state of materials coated onto the sensor chip, and ifan antigen or an antibody is flowed onto a chip coated with the antigenor antibody, it causes changes in refractive index due to their bindingand Kd values are thus calculated from the measured values.

First, anti-His antibody was immobilized on a CM5 sensor chip (GEhealthcare) up to 8,000 RU levels using a pH 5.0 acetate solution and anamine coupling kit (GE Healthcare). 6 μg/ml of a recombinant hAng2(C-His, R&D Systems) protein was flowed onto the chip to be captured at100 to 200 RU levels. The antibody obtained in Example 2 above wasdiluted serially to twice each time starting from 100 nM concentrationand it was each flowed onto the chip to allow it to be bound to (on),dissociated from (off), and regenerated (using 10 mM NaOH solution) fromthe antigen captured on the sensor chip, thereby to measureantigen-antibody affinity. With regard to hAng2, such experiments wereconducted, and the results are as shown in the following Table 3.

TABLE 3 Antibody Name hAng2 (Kd) SAIT-ANG2-AB-m10D6 8.0 nM

2.2. Gene Cloning of Anti-Ang2 Antibody

A whole RNA was obtained using RNeasy mini kit (Qiagen) from theantibody-producing hybridoma (2×10⁶ cells) obtained from Example 2.1above. Then, by using this as a template, only the gene sequence of theheavy chain and light chain variable regions of the monoclonal antibodyto be produced in the hybridoma was amplified using a OneStep RT-PCR kit(Qiagen), a Mouse Ig-Primer Set (Novagen), and a thermocycler (GeneAmpPCR System 9700, Applied Biosystem) under the following conditions: 5min. at 94° C.; [30 min. at 50° C., 15 min. at 95° C.], [1 min. at 94°C., 1 min. at 50° C., 2 min. at 72° C.]×35 cycles; 6 min. at 72° C.;cooling to 4° C.

The PCR products obtained from each reaction were subjected to a directDNA sequencing to obtain the CDR, heavy chain variable regions and lightchain variable regions of the antibody, and nucleotide sequencesencoding them, and the obtained results are set forth in the followingTables 4 to 7.

TABLE 4 Antibody Heavy Chain CDR Name CDRH1-KABAT CDRH2-KABATCDRH3-KABAT SAIT-ANG2- SDYAWN YINYSGNTDYN GNFEGAMDY AB-m10D6 (SEQ ID NO:PSLKS (SEQ ID NO: 1) (SEQ ID NO: 2) 3)

TABLE 5 Antibody Light Chain CDR Name CDRL1-KABAT CDRL2-KABATCDRL3-KABAT SAIT-ANG2- KASQSVSNDVA YASNRYP QQDYSSPWT AB-m10D6(SEQ ID NO: (SEQ ID NO: (SEQ ID NO: 4) 5) 6)

TABLE 6 Antibody Name Heavy Chain Variable Region Sequence SAIT-ANG2-DVQLQESGPGLVKPSQSLSLTCTVTGYSIT SDYAWN AB-m10D6 WIRQFPGNKLEWMGYINYSGNTDYNPSLKS RSSITR DTSKNQFFLQLNSVTTGDTATYYCAR GNFEGAMDY WGQGTSVTVSS (SEQ ID NO: 7) GATGTGCAGCTTCAGGAGTCGGGACCTGACCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATAAACTACAGTGGTAACACTGACTACAACCCATCTCTCAAAAGTCGAAGCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGGGGACACAGCCACATATTACTGTGCAAGAGGTAACTTCGAAGGTGCTATGGACTACTGG GGTCAAGGAACCTCAGTCACCGTCTCCTCA(SEQ ID NO: 8)

TABLE 7 Antibody Name Light Chain Variable Region Sequence SAIT-ANG2-SIVMTQTPKFLLVSAGDRVTITC KASQSVSNDVA WY AB-m10D6 QQKPGQSPKLLIY YASNRYPGVPDRFTGSGYGTDFT FTISTVQAEDLAVYFC QQDYSSPWT FGGGTKLEIK (SEQ ID NO: 9)agtattgtgatgacccagactcccaaattcctgcttgtatcagcaggagacagggttaccataacctgcaaggccagtcagagtgtgagtaatgatgtagcttggtaccaacagaagccagggcagtctcctaaactgctgatatactatgcatccaatcgctaccctggagtccctgatcgcttcactggcagtggatatgggacggatttcactttcaccatcagcactgtgcaggctgaagacctggca gtttatttctgtcagcaggattatagctctccgtggacgttcggtggaggcaccaagctggaaatcaaa (SEQ ID NO: 10)

(In above Tables 6 and 7, underlined bold letters are CDR1, CDR2, andCDR3 in sequence)

Example 3 Competition ELISA Assay of Antibody 10D6 against Ang2-Tie2Binding

Ang2-Tie2 binding competition ELISA was conducted using the antibodybinding to Ang2 prepared in Example 2-1 above.

More specifically, MaxiSorp™ flat-bottom plate (Nunc) of 96-well wascoated with hTie2-Fc (R&D Systems) which is a protein bound with 4 ug(microgram)/ml of Fc of human IgG1. After that, the plate was washedfive times with 0.05% (v/v) Tween-20-containing PBS (phosphate buffersaline) and then blocked with 1% (v/v) BSA (bovine serum albumin;Sigma)-containing PBS at a room temperature for 2 hour.

For Ang2:Tie2 competition ELISA, each anti-Ang2 antibody obtained inExample 2 was placed at various concentrations of 400 nM to 0.001 nMinto each well coated with the hTie-2/Fc fusion protein along with 1%(v/v) BSA and 400 ng/ml of a FLAG-tagged hAng2 and then, the plate wasallowed to react at a room temperature for 2 hours and washed five timeswith PBST. After that, an anti-FLAG antibody (Sigma) conjugated with HRPdiluted in 1% (v/v) BSA-containing PBS at a ratio of 1:5,000 (v/v) wasadded in an amount of 100 ul (microliter) to each well to react at aroom temperature for 1 hour and then, the plate was washed five timeswith PBST. Lastly, 100 ul (microliter) of TMB substrate (Cell Signaling)was added to each well of the plate to induce color development for 3min. and then, the reaction was ceased by the addition of 100 ul of Stopsolution (Cell Signaling) and OD450 values were measured on a platereader (Molecular Devices).

For comparison, the same test was carried out using 4H10 which is ananti-Ang2 antibody inhibiting Ang2-Tie2 binding. The 4H10 is an antibodyhaving the following heavy chain variable region and light chainvariable region.

Heavy chain variable region (SEQ ID NO: 12):EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYDMS WVRQAPGKGLEWVS L IS PDSSSIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKDL ISFWRGGFDY WGQGTLVTVSSLight chain variable region (SEQ ID NO: 13) QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVN WYQQLPGTAPKLLIY ADS NRPS GVPDRFSGSKSGTSASLAISGLRSEDEADYYCGSWDYSLSG YV FGGGTKLTVLG

An inhibitory degree (%) against Ang2-Tie2 binding is shown in FIG. 1.As seen in FIG. 1, unlike 4H10 which is an anti-Ang2 antibody inhibitingAng2-Tie2 binding, antibody 10D6 did not inhibit binding betweenAng2-Tie2 receptor.

Example 4 Verification of Antigen Recognizing Site (Epitope) of Ang2Antibody

To verify the epitope (or specific binding site) of the anti-Ang2antibody prepared in Example 2, an ELISA was performed using arecombinant protein where a receptor binding domain (RBD) of Ang2protein in the form of being tagged with Flag was mutated by artificialmeans.

Each well of a 96-well MaxiSorp™ flat-bottom plate (Nunc) was coatedwith 50 ul (microliter) of 1000 nM antibody. Then, the plate was washedfive times with 0.05% (v/v) Tween-20-containing PBS (PBST) and blockedwith 1% (v/v) BSA-containing PBS at a room temperature for 2 hours. 250ng of each mutant Ang2 protein obtained by substituting S417, Q418,P419, N421, I434, D448, A449, P452, Y460, N467, K468, or F469 residue ofAng2 protein tagged with a FLAG sequence (DYKDDDDK, Sigma) at itsN-terminal with alanine was added to each well of the plate, which wasthen allowed to react at a room temperature for 2 hours.

The plate was washed five times with 0.05% (v/v) Tween-20 containingPBS, reacted with an anti-FLAG antibody (SIGMA) conjugated with HRPwhich was diluted in 1% (v/v) BSA-containing PBS at a ratio of 1:5,000(v/v) at a room temperature for 1 hour, and washed five times with 0.1%(v/v) Tween-20-containing PBS.

Finally, 50 ul of TMB substrates (Cell signaling) was added to each wellof the plate to induce color development at a room temperature for 3min. and the reaction was ceased by the addition of 50 ul of Stopsolution (Cell signaling) and then, OD450 values were measured on aplate reader (Molecular Devices). By comparing binding affinities withmutated Ang2 to those of unmutated Ang2, each epitope of Ang2 antibodieswas identified. The thus obtained measurement results of the bindingaffinities (%) with mutant Ang2 against the binding affinity with thenative Ang2 are shown in the following Table 8.

TABLE 8 Loop 1 Loop 2 Loop 3 Loop 4 417 418 419 421 434 448 449 452 460467 468 469 10D6 101.17 38.94 41.08 109.09 109.49 104.55 97.86 102.15103.15 106.84 110.27 108.79

Example 5 Phosphorylation Induction of Tie2 Receptor by Antibody 10D6

As Ang2 induces a change in vascular endothelial cells by binding to aTie-2 receptor expressed in the vascular endothelial cells to induce thephosphorylation of the receptor and activate it, a test for analyzing aninfluence of the anti-Ang2 antibody on Tie2 phosphorylation wasconducted using a cell-based assay. For this, HUVEC (ATCC) cells (1×10⁶)were cultured in a 100 mm culture dish using EGM-2 (Lonza) media at 37°C. and when they reached 80˜90% confluency, the media were replaced withserum-free media and cultured at 37° C. for 6 to 16 hours. The dish waswashed once with PBS and after the replacement with 1 nM sodiumorthovanadate (Sigma)-mixed serum free media (Lonza), they were furthercultured for 10 min. After washed once again with PBS, the culturedcells were treated with a mixture prepared by mixing the anti-Ang2antibody (10D6) having various concentrations (600˜0.06 nM) with 40 nMof Ang2 protein (R&D systems) and letting them stand for 20 min. andfurther cultured for 10 min. The cells were washed using PBS, treatedwith 400 ul of a lysis buffer (Roche), collected to a tube to bedissolved at 4° C. for 30 min. and then, centrifuged at 13,000 rpm for15 min. to measure a supernatant using Nanodrop.

1 ug of Tie2 antibody (R&D system) was added to 0.8 mg of a cell lysate,which was then overnight reacted at 4° C. and then subjected toimmunoprecipitation by the addition of protein A bead (GE Healthcare)thereto. The thus obtained reactant was centrifuged at 13,000 rpm for 15min. to obtain a pellet, which was washed two to three times with alysis buffer (Roche), added to a sample buffer (Invitrogen) mixed with areducing agent, and boiled at 95° C. for 5 min., and then, applied toNuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) and transferred ontoNitrocellulose membrane (Invitrogen).

To see the presence of the phosphorylation of Tie2, the membranes wereblocked with PBST mixed with 3% (v/v) skim milk (Sigma) for 30 min. andidentified using an HRP-conjugated anti-phospho tyrosine antibody(Millipore). For Tie2 identification, the blots were reacted in astripping buffer (Thermo) for 15 min, then blocked again and identifiedusing an Tie2 antibody (Santa cruz).

The thus obtained results are shown in FIG. 2A. As shown in FIG. 2A,when antibody 10D6 was added together with Ang2, the phosphorylation ofTie2 was more strongly induced at every antibody concentration rangetested than the case in which Ang2 was treated alone.

In addition, referring to the above method, when 60 nM of antibody 10D6and Ang2 were treated, the phosphorylation level of Tie2 was measuredand compared with the case of treating a control antibody (anti-Ang2antibody (Regeneron) which has MOA of inhibiting binding between Ang2and Tie2; represented as ‘RG antibody’). The obtained immunoblottingresults are shown in FIG. 2 b. After treating with the antibody (10D6 orRG), the blot band density of pTyr and Tie2 was measured using ImageJsoftware and pTyr/Tie2 ratio was calculated. The obtained result isdemonstrated in FIG. 2C.

In FIGS. 2B and 2C, “NC” represents Tie2 phosphorylation results in agroup which is not treated with antibody nor Ang2. As shown in FIGS. 2Band 2C, when treated with antibody 10D6, Tie2 phosphorylation level isincreased by 180% compared to the case that Ang2 is treated only withouttreating with antibody; whereas, when treated with control antibody RG,Tie2 phosphorylation level is decreased by 67% compared to the case thatAng2 is treated only without treating with antibody, indicating thatantibody 10D6 has about 8.6-fold higher Tie2 phosphorylation effect thancontrol antibody.

Example 6 Activation Induction of Tie2 Signaling by Antibody 10D6

To see whether antibody 10D6 induces the activation of the downstreamsignaling of a Tie2 receptor as well as the activation of the Tie2receptor itself, the phosphorylation degrees of proteins participatingin the downstream signaling when Ang2 alone or Ang2 and antibody 10D6was treated were tested using immune blotting. To compare the activationdegrees of the downstream signaling, the same test was conducted withregard to a group in which Ang1 (R&D systems), and Ang2 (R&D systems)and an anti-Ang2 antibody (RG antibody; control antibody having MOAinhibiting Ang2-Tie2 binding, Regeneron Co.) were treated together.

Specifically, HUVEC (ATCC) cells (1×10⁶) were cultured in a 6-wellculture dish using EGM-2 (Lonza) media at 37° C. and when they reached80˜90% confluency, the media were replaced with serum-free media (Lonza)and cultured at 37° C. for 6 to 16 hours. The dish was washed once withPBS, and the cultured cells were treated with a mixture prepared bymixing 60 nM of the Ang2 antibody (10D6) with 40 nM of Ang2 protein (R&Dsystems) and letting them stand for 20 min. and further cultured for 30min. For comparison, groups in which Ang1 (R&D systems) 4 nM, Ang2 (R&Dsystems) 40 nM, and Ang2 (R&D systems) 40 nM+anti-Ang2 antibody(Regeneron) 60 nM were treated respectively were prepared.

The cells were washed using PBS, treated with a lysis buffer (Roche),collected to a tube to be dissolved at 4° C. for 30 min. and then,centrifuged at 13,000 rpm for 15 min. to measure a supernatant. A samplebuffer (Invitrogen) mixed with a reducing agent was added to 25 ug of acell lysate, which was boiled at 95° C. for 5 min., and then, applied toNuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) and transferred ontoNitrocellulose membrane (Invitrogen).

To see the presence of the phosphorylation of Akt, eNOS and 42/44involved in the downstream signaling, the blots were blocked with PBSTmixed with 3% (v/v) skim milk (Sigma) for 30 min. and then treated withan anti pAkt antibody, anti p-eNOS antibody, and anti p-42/44 antibody(all of them; cell signaling). The blots were reacted in a strippingbuffer (Thermo) for 15 min. and then blocked again to identify Akt,eNOS, and 42/44 using an anti Akt antibody, anti eNOS antibody, and anti42/44 antibody (all of them; cell signaling).

The thus obtained results are shown in FIG. 3A. As seen in FIG. 3A, inthe Ang1 sole-treatment group and the Ang2 and 10D6 co-treatment group,downstream signaling was strongly induced in comparison with the Ang2sole-treatment group and the Ang2 and RG antibody co-treatment group,and the effects in the Ang2 and antibody 10D6 co-treatment group was atleast equal to those in the Ang1 sole-treatment group.

In addition, the phosphorylation of Tie2 receptor and a protein (Akt)which participates in downstream signaling of Tie2 by treating anti-Ang2antibody was measured in animal model (in vivo). More particularly, 5mg/kg of antibody was injected alone or together with 20 ug of Ang2 intotail vein of 7-8 week old C57BL6 mouse, and 1 hour after, lung tissuewas removed. The obtained lung tissue was subjected to homogenizationlysis using lysis buffer (Roche) and FastPrep kit (MP biomedicals).Activities of Tie2 and Akt in the obtained tissue lysate were measuredby the above-described method.

The obtained results are demonstrated in FIGS. 3B and 3C. In FIGS. 3Band 3C, “REGN” or “RG” represents a control antibody (anti-Ang2 antibodyof Regeneron). As shown in FIGS. 3B and 3C, even in vivoexperimentation, antibody 10D6 exhibits considerable effect ofphosphorylating Tie2 and a protein, Akt, participating in downstreamsignaling of Tie2.

Example 7 ELISA Assay for Identifying Formation of 10D6-Ang2-Tie2Complex

As it was confirmed that 10D6 anti-Ang2 antibody activates Tie2signaling without inhibiting Ang2-Tie2 binding, an ELISA was conductedto see whether a complex between the antibody and Ang2:Tie2 receptor isformed.

A 96-well MaxiSorp™ flat-bottom plate (Nunc) was coated with 4 ug/ml ofTie2-Fc (R&D systems) or BSA (Sigma). Then, the plate was washed fivetimes with 0.05% (v/v) Tween-20-containing PBS (Phosphate Buffer Saline)and blocked with 1% (v/v) BSA (Bovine serum albumin; Sigma)-containingPBS at a room temperature for 2 hours. 0.25 ug/ml of Ang2 and 2 ug/ml ofantibody 10D6 were added to each well of the plate, which was allowed toreact at a room temperature for 2 hours and then washed five times withPBST. After that, an anti-mouse IgG antibody (Sigma) conjugated with HRPdiluted in 1% (v/v) BSA-containing PBS at a ratio of 1:5,000 (v/v) wasadded in an amount of 100 ul to each well to react at a room temperaturefor 1 hour and then, the plate was washed five times with PBST. Lastly,100 ul (microliter) of TMB substrate (Cell Signaling) was added to eachwell of the plate to induce color development for 3 min. and then, thereaction was ceased by the addition of 100 ul of Stop solution (CellSignaling) and OD450 values were measured on a plate reader (MolecularDevices).

The thus obtained results are shown in FIG. 4. As seen in FIG. 4, it wasconfirmed that antibody 10D6 formed a complex by binding to Ang2 whichwas bound to Tie2.

Example 8 Activation Induction via Tie2 Receptor Clustering byDimerization of Antibody 10D6

To see whether the activation of a Tie2 receptor is resulted from thedimerization of antibody 10D6, Fab fragments of antibody 10D6 werepurified and then used for this experiment. The Fab fragments wereobtained from antibody 10D6 using an Fab Preparation Kit (Pierce). As adigestion buffer, PBS containing 20 mM EDTA and 20 mM L-Cysteine wasused, and the antibody was separated into Fab and Fc by the treatmentwith an immobilized papain at a room temperature for 2 hours. The Fcfragments were removed from the reaction solution using MabSelectSuRecolumn (GE Healthcare) to isolate and purify pure Fab fragments only.

HUVEC (ATCC) cells (1×10⁶) were cultured in a 100 mm culture dish usingEGM-2 (Lonza) media at 37° C. and when they reached 80·90% confluency,the media were replaced with serum-free media (Lonza) and cultured at37° C. for 6 to 16 hours. The dish was washed once with PBS and afterthe replacement with 1 nM sodium orthovanadate (Sigma)-mixed serum freemedia (Lonza), they were further cultured for 10 min. After washed oncewith PBS, the cultured cells were treated with a mixture prepared bymixing 60 nM of 10D6 Ang2 antibody or 10D6 Fab with 40 nM of Ang2protein (R&D systems) and letting them stand for 20 min. and furthercultured for 10 min. The cells were washed using PBS, treated with 400ul of a lysis buffer (Roche), collected to a tube to be dissolved at 4°C. for 30 min. and then, centrifuged at 13,000 rpm for 15 min. tomeasure a supernatant using Nanodrop.

1 ug of Tie2 antibody (R&D system) was added to 0.8 mg of a cell lysate,which was then overnight reacted at 4° C. and then subjected toimmunoprecipitation by the addition of protein A bead (GE Healthcare)thereto. The thus obtained reactant was centrifuged at 13,000 rpm for 15min. to obtain a pellet, which was washed two to three times with alysis buffer (Roche), added to a sample buffer (Invitrogen) mixed with areducing agent, and boiled at 95° C. for 5 min., and then, applied toNuPAGE Novex 4-12% Bis-Tris gel (Invitrogen) and transferred ontoNitrocellulose membrane (Invitrogen).

To see the presence of the phosphorylation of Tie2, the membranes wereblocked with PBST mixed with 3% (v/v) skim milk (Sigma) for 30 min. andidentified using an HRP-conjugated anti-phospho tyrosine antibody(Millipore). For Tie2 identification, the blots were reacted in astripping buffer (Thermo) for 15 min, then blocked again and identifiedusing an Tie2 antibody (Santa cruz).

To see the presence of the phosphorylation of Akt, eNOS and 42/44involved in the downstream signaling, the blots were blocked with PBSTmixed with 3% (v/v) skim milk (Sigma) for 30 min. and then treated withan anti pAkt antibody, anti p-eNOS antibody, and anti p-42/44 antibody(all of them; cell signaling). The blots were reacted in a strippingbuffer (Thermo) for 15 min. and then blocked again to identify Akt, and42/44 using an anti Akt antibody, and anti 42/44 antibody (all of them;cell signaling).

The thus obtained results are shown in FIG. 5. As seen in FIG. 5, whenantibody 10D6 was added together with Ang2, the phosphorylation of Tie2and Akt and 42/44 occurred whereas when the 10D6 Fab was treated,activation was not observed. Therefore, it was confirmed that the Tie2receptor was clustered by the dimerization of the 10D6 antibodies andstrongly phosphorylated.

Example 9 Internalization of Tie2 Receptor by Antibody 10D6

HUVEC (ATCC) cells (1×10⁶) were cultured in m-slide 8 wells (#80826,ibidi) for 24 hours and then subjected to serum starvation in serum-freeEBM-2 media (Lonza) for 3 hours. 200 ng/ml of Ang1 (R&D systems) or 2ug/ml of Ang2 (R&D systems) and 10 ug/ml of antibody 10D6 were eachdiluted in EBM-2 media and then, the cells were treated with them andincubated according to time specified in the picture. The cells fixedwith 4% formaldehyde (Sigma) was dyed first with an anti-Tie2 antibody(R&D systems) and then subjected to a secondary dye with an anti-goatAlexa 555 antibody (red, Life technology) and an anti-mouse Alexa 488antibody (green, Life technology). The dyed cells were treated withVectashield mounting medium with DAPI (Vector labs) and then observedusing a confocal laser scanning microscopy (Carl Zeiss).

The results are shown in FIG. 6A (red: Tie2, blue: nucleus, green; 10D6)and 6B. As seen in FIG. 6 a, when Ang2 and antibody 10D6 were treated,the internalization of the Tie2 receptor started from 30 min., like Tie2endocytosis occurring when treated with Ang1 (dots in the expandedpictures). Also, it was confirmed that the internalized Tie2 andantibody 10D6 were located in an early endosome which is anintracellular organelle, through anti-EEA1 antibody (Cell Signaling) dyein FIG. 6 b.

Example 10 Cell Growth and Mobility Increase by Antibody 10D6

The measurement assay of the proliferation of vascular and lymphaticendothelial cells was conducted using a BrdU assay kit (Roche) Cell.Vascular endothelial cells of P3˜P8 (HUVEC, ATCC) or lymphaticendothelial cells (Lonza) were placed in a collagen-coated 96-well plate(BD Bioscience) at 3000˜5000 cells/well and cultured in EGM-2 media(Lonza) for 16˜24 hours. A mixture of 2 ug/ml of Ang2 (R&D systems) and10 ug/ml of antibody 10D6 in 2% EBM media was added to the 96-well platewashed with PBS, which was then cultured for 3 days.

For efficiency comparison, an anti-Ang2 antibody (MEDI: Medlmmune Co.)was used. For a BrdU assay, 10 ul of BrdU solution diluted at 1:1,000was added to each well of the plate to label the cells. The cells werecultured for 1 hour and after the removal of the culture solution, 100ul of Fixation/Denaturation solution (Roche) was added to each well andthen, removed after 30-min wait. An anti-BrdU antibody (Roche)conjugated with peroxidase was diluted in a dilution buffer (Roche) at1:100, added to each well to react for 1 hour and then, washed fourtimes using a washing buffer (Roche). 100 ul of a TMB substrate wasadded thereto to react and then, absorption was measured at 450 nm usinga Microplate reader (Perkin Elmer).

The thus obtained results are shown in FIGS. 7A and 7B. As seen in FIGS.7A and 7B, the growth of cells was increased in the group in which Ang2was added together withantibody 10D6 in both vascular endothelial cellsand lymphatic endothelial cells, and in the case of MEDI antibody, thegrowth of the cells by Ang2 was decreased.

Also, the measurement of the mobility of lymphatic endothelial cells wasconducted using xCelligence RTCA (Realtime cell analyzer; GEHealthcare). The RTCA is a non-invasive cell monitoring system bymeasuring impedance in real time to identify the change of a cell. Toperform a cell migration assay, a CIM-plate16 (GE Healthcare) with alower chamber and an upper chamber was used wherein microelectrodes formeasuring impedance are arranged in the upper chamber and if the cellsseeded into the chamber migrate through fine holes, the migration degreeof the cells can be identified through their adhesion to themicroelectrodes, and it was referred to as migration index. Thelymphatic endothelial cells (P5-7; Lonza) which grew up in EGM-2 media(Lonza) were cultured in 1% FBS-added EBM media for 6 hours. 2 ug/ml ofAng2 and antibody 10D6 in 2% FBS-added EBM media were added to each wellof the lower chamber of the CIM-plate 16, which was assembled with theupper chamber coated with fibronectin (Sigma). For efficiencycomparison, an anti-Ang2 antibody (MedImmune Co.) was used. 30 ul ofserum-free EBM media was each added to the upper chamber and then, forequilibration between the plate and media, while being placed in anincubator for 1 hour, the CIM-plate was installed on a device station inthe incubator and then, background values were measured. The lymphaticendothelial cells re-suspended with serum-free media were seeded in anamount of 60,000 cells/well, allowed to settle down for 15 min. andthen, installed on the device to measure cell migration in real time.The cell migration degree was shown as slope (1/hr).

The thus obtained results are shown in FIG. 8. As seen in FIG. 8, whilethe migration of the lymphatic endothelial cells to which Ang2 was addedtogether with antibody 10D6 was increased, the migration of the cells byAng2 was inhibited in the case of MEDI antibody.

Example 11 Cell Permeability Suppression by Antibody 10D6

To see the cell permeability suppression effects of antibody 10D6, invitro vascular permeability assay kit (Millipore) was employed. HUVEC(ATCC) cells (5×10⁴) were added to a collagen-coated transwell andcultured for 2 days to prepare a confluent monolayer. It was treatedwith 200 ng/ml of Ang2 or 200 ng/ml of Ang1 (R&D systems) alone, or 200ng/ml of Ang2 and 1 ug/ml of antibody 10D6, pre-incubated for 30 min.and then, treated with 100 ng/ml of LPS (Lipopolysaccharide; Sigma) or100 ng/ml TNF-a (R&D systems) to induce the permeability of HUVECmonolayer. LPS or TNF-a weakens binding between HUVEC cells to form agap therebetween, thereby increasing the migration of materials in theHUVEC monolayer. After 6-hour incubation for the LPS treatment group and22-hour incubation for the TNF-a group, the upper chamber was treatedwith FITC-dextran (Millipore) and incubated for 18 min. The fluorescenceof FITC-dextran migrated to the lower chamber was measured at 485/535 nm(Ex/Em) setting using an Envision 2104 multilabel Reader (Perkinelmer).

The thus obtained results are shown in FIG. 9. FIG. 9 is a graph showingthe strength of fluorescence measured above as relative folds, referringto the vascular permeability induced by LPS or TNF-a. Through theresults, it was confirmed that the vascular permeability increased byLPS or TNF-a was decreased to the control level in the Ang1sole-treatment group and in the Ang2 and 10D6 co-treatment group.

Example 12 Anti-Inflammatory Effect by Antibody 10D6

To see anti-inflammatory effects by antibody 10D6, HUVEC (ATCC) cells(4×10⁵) were treated with Ang2 alone or Ang2 together with antibody10D6, and a group in which Ang1 and Ang2 (R&D systems) and Regeneron Co.antibody (RG antibody) were co-treated was included as a control group.HUVEC cells were treated with 0.2 ug/ml of Ang1, 0.1 ug/ml of Ang2, or0.1 ug/ml of Ang2 and 0.5 ug/ml of antibody, respectively and after30-min. pre-incubation, they were treated with 100 ng/ml of LPS (Sigma)to induce inflammatory response and incubated for 5 hours.

The degree of the inflammatory response was identified by measuring themRNA expression degree of an inflammatory adhesion substance (ICAM-1,E-selectin). The cells were washed using PBS, RNA was extracted usingRNeasy Mini kit (Qiagen), and among them, 2 ug of RNA was synthesizedinto cDNA using Transcriptor First Strand cDNA synthesis kit (Roche).qPCR reaction was performed using RT2 SYBR™ Green Mastermix (Qiagen) andLightCycler™ 480 Real-Time PCR System (Roche). As an internal controlfor calibrating the amount of RNA in a sample, HPRT1 was used, and qPCRcomplied with the following procedures with regard to all the primers.

Step 1: 95° C., 10 min; Step 2 (45 cycles): Step 2-1: 95° C., 15 sec;Step 2-2: 60° C., 1 min; Step 3: 65° C., 15 sec; Step 4: 95° C.,continuous (every 20° C.); Step 6: 40° C., 10 sec.

The primer sequences used in qPCR are set forth in the following Table9.

Step 1: 95° C., 10 min; Step 2 (45 cycles): Step 2-1: 95° C., 15 sec;Step 2-2: 60° C., 1 min; Step 3: 65° C., 15 sec; Step 4: 95° C.,continuous (every 20° C.); Step 6: 40° C., 10 sec.

The primer sequences used in qPCR are summarized in Table 9 as follows:

TABLE 9 Representative Gene PCR primer sequence (5′ -> 3′) Public IDSymbol sense antisense NM_000201.2 ICAM-1 ccttcctcaccgtgtactggaacctcagcctcgctatgg NM_000450.2 E-selectin accagcccaggttgaatgggtggacaaggctgtgc NM_000194.2 HPRT1 tgaccttgatttattttgcatacccgagcaagacgttcagtcct

The relative expression amounts of ICAM-1 and E-selectin measured aboveare shown in FIG. 10. As seen in FIG. 10, the expression of ICAM-1 andE-selectin induced by LPS were remarkably suppressed in the Ang1sole-treatment group and in the Ang2 and antibody 10D6 co-treatmentgroup.

Meanwhile, if the expression of inflammatory adhesion substances(ICAM-1, E-selectin, etc.) of vascular endothelial cells is increased,binding between the inflammatory cells and the vascular endothelialcells are accelerated and inflammatory response is thus induced. Toconfirm this, an experiment for measuring binding between inflammatorycells HL-60 (ATCC) and HUVEC was conducted.

HUVEC cells (4×10⁵) were cultured in a 24-mutiwell plate and treatedwith 0.1 ug/ml of Ang2 alone or 0.1 ug/ml of Ang2 together with 0.5ug/ml of antibody 10D6, and a group in which 0.2 ug/ml of Ang1, and 0.1ug/ml of Ang2 (R&D systems) and 0.5 ug/ml of Regeneron Co. antibody (RGantibody) were co-treated was included as a control group. As above, theHUVEC cells were treated with Angiopoietin and antibody and after30-min. pre-incubation, they were treated with 1 ng/ml of TNF-a(eBioscience) to induce inflammatory response and incubated for 2 hoursand 30 min.

After the HUVEC cells were washed with complete media (Lonza), HL-60cells dyed with Calcein-AM (BD bioscience) solution were added at theamount of 2.5×10⁵ cells to each well and incubated for 1 hour. To removethe HL-60 cells not bound to the HUVEC cells, the cells were washedthree times using PBS and treated with a lysis buffer (0.1% SDS, 50 mMTris.HCl, pH 8.0). The obtained lysate was transported to a 96-wellplate, and fluorescence strengths derived from the HL-60 cells weremeasured at 485/535 nm (Ex/Em) setting using an Envision 2104 multilabelReader (Perkin Elmer).

The thus obtained results are shown in FIG. 11. The graph of FIG. 11illustrates the strengths of fluorescence measured above as relativefolds and it is proportional to the number of the HL-60 cells adhered tothe HUVEC cells. Through the results, binding between HUVEC cells andHL-60 cells induced by TNF-a was remarkably suppressed in the Ang1sole-treatment group and in the Ang2 and antibody 10D6 co-treatmentgroup.

Example 13 Colo205 Tumor Growth Inhibitory Effect of Anti-Ang2 Antibody

To see the tumor growth inhibitory effects of Ang2 antibody, acolorectal cancer xenograft model using a human colorectal cancer cellline Colo205 (ATCC) was employed.

The Colo205 cell line was cultured using 10% FBS (Gibco)-supplementedRPMI-1640 (Gibco) media. 5×10⁵ of Colo205 cell line were re-suspended in100 ul of serum-free media and then subcutaneously injected intoanesthetic 4˜5-week-old BALB/c nude mice (Shanghai SLAC LaboratoryAnimal Co. Ltd.) anesthetized with 1˜2% isoflurane. When the size oftumors reached 100˜200 mm³, anti-Ang2 antibody (10D6) was injected atthe concentration of 10 mg/kg via an intraperitoneal injection twice aweek and the size of the tumors was measured.

The size of tumor (V) was calculated using the following formula:

V=(length×width²)/2

The thus obtained results are shown in FIG. 12. The X-axis of FIG. 12indicates days on which the antibody was treated, as days aftergrouping. As in FIG. 12, antibody 10D6 inhibited the growth ofcolorectal cancer.

Example 14 LLC Tumor Growth Inhibitory Effect of Anti-Ang2 Antibody

To see the tumor growth inhibitory effects of Ang2 antibody, acolorectal cancer Xenograft model using a LLC (Lewis Lung Carcinoma) wasemployed.

LLC cells (ATCC) were cultured using 10% FBS (Gibco)-supplemented DMEM(Gibco) media. 1×10⁶ of LLC cells were re-suspended in 100 ul of PBS(Gibco) and then subcutaneously injected into 6˜8-week-old C57BL/6 mice(Jackson Laboratory) anesthetized with 1˜2% isoflurane.

When the size of tumors reached 50˜100 mm³, anti-Ang2 antibody (10D6)was injected at the concentration of 10 mg/kg via an intraperitonealinjection three times a week and the size of the tumors was measured.

The size of tumor (V) was calculated using the following formula:

Volume (V)=(width)₂×length/2

For comparison, the same experiment was performed to a control grouptreated with Fc of IgG1 and a comparative group treated with antibody4H10 (refer to Example 3).

The thus obtained results are shown in FIG. 13. As shown in FIG. 13,antibody 10D6 that induces binding between Ang2 and Tie2 inhibits thegrowth of the tumor by about 40% compared to the control group. Suchtumor growth inhibition effect of antibody 10D6 can be considered asbeing significant, compared the fact that antibody 4H10 that inhibitsbinding between Ang2 and Tie2 inhibits the growth of the tumor by about27%.

Example 15 Vascular Normalization Effect of Anti-Ang2 Antibody

To examine changes in blood vessels in tumor tissues after treated withantibody, frozen sections were obtained and subjected toimmunofluorescence staining using a blood vessel specific marker, CD31and a pericyte specific marker, PDGFR-β.

In particular, the tumor tissues obtained from Example 14 were frozenusing OCT compound (Sakura Finetek), and frozen sections were obtainedtherefrom using cryostat (Leica). The obtained tumor frozen sectionswere blocked with 0.3% TBST (TBS mixed with 0.3% Triton X-100) buffersupplemented with 5% goat serum (Jackson Immunoresearch) for one hour.To the blocking buffer, hamster anti-CD31 antibody (1:200, Millipore)and rat anti-PDGF receptor beta antibody (1:200, eBioscience) wereadded, and the frozen sections were added thereto to be allowed to reactfor 3 hours. the reacted product was washed with TBS three times, andthen, the blocking buffer mixed with cy3-conjugated anti-hamster IgGantibody and FITC-conjugated anti-rat IgG antibody (1:1,000, JacksonImmunoresearch) was added the frozen sections to be allowed to react for2 hours. After washing with TBS three times, the frozen sections werecovered with coverslip (Marenfield) using Fluorescence mounting medium(DAKO). The stained tumor frozen sections were observed using LSM5(Zeiss) confocal microscope. For comparison, the same experiment wasperformed using Fc of IgG1 and antibody 4H10 (refer to Example 3).

The obtained results were illustrated in FIG. 14. In FIG. 14, redindicates CD31+ area and green indicates PDGFR-beta+area. As shown inFIG. 14, compared to Fc-treated tumors or 4H10-treated tumors, in10D6-treated tumors, it is observed that most of blood vessels in tumorsare decreased and normalized. For example, compared to the control andcomparative groups, in 10D6 treated group, the shapes of the bloodvessels has more regular shapes and diameters and more decreasedsprouting. In addition, the characteristics of vascular normalization intumor tissues, such as close association of the PDGFR-beta positivepericytes around blood vessels and the like, were observed. Such resultsindicate that antibody 10D6 decreases the density of blood vessels intumor and induces vascular normalization.

Example 16 Effect of Anti-Ang2 Antibody of Increasing Functions of TumorBlood Vessels

To see the functions of tumor blood vessels treated with antibody 10D6,perfusion level of the blood vessel was examined by Lectin perfusionassay. Referring to the method described in Example 14, LLC tumor cellswere subcutaneously injected into mice. When the size of tumors reached50˜100 mm³, anti-Ang2 antibody 10D6 was injected at the concentration of10 mg/kg via an intraperitoneal injection three times a week and thesize of the tumors was measured. For comparison, the same experiment wasperformed to a control group treated with Fc of IgG1 and a comparativegroup treated with antibody 4H10 (refer to Example 3).

After administering the antibody five-times, the mice were injected withFITC-Lectin (Vector laboratory) via tail vein, and after 20 minutes,subjected to perfusion fixation. Tumor frozen sections were obtained andimmunostained using hamster anti-CD31 antibody (1:200, Millipore).Images were obtained using LSM5 confocal microscope (Zeiss), and Lectin+area/CD31+ area was quantified using ImageJ software(http://rsb.info.nih.gov/ij).

The obtained results are illustrated in FIGS. 15A and 15B. As shown inFIGS. 15A and 15B, compared to Fc-treated or 4H10-treated group, in 10D6treated group, the perfusion function of tumor blood vessels is enhancedand the percentage of Lectin+ area (green) in CD31+ area (red) isincreased.

Example 17 Effect of Anti-Ang2 Antibody of Decreasing Necrosis Inside ofTumor

To see the effects of the structurally and functionally normalized bloodvessels in tumor by treatment of antibody 10D6 on the tumor tissues,referring to the method of Example 14, LLC tumor cells weresubcutaneously injected into mice. When the size of tumors reached50˜100 mm³, anti-Ang2 antibody 10D6 was injected at the concentration of10 mg/kg via an intraperitoneal injection three times a week and thesize of the tumors was measured. After administering the antibodyfive-times, the tumor sections were stained with H&E according tostandard protocol, and then, the Eosin+ cell necrotic region in tumortissues was confirmed. For comparison, the same experiment was performedto a control group treated with Fc of IgG1 and a comparative grouptreated with antibody 4H10 (refer to Example 3).

The obtained results are illustrated in FIG. 16. As shown in FIG. 16, in10D6-treated group, smaller cell necrotic region is observed, comparedto Fc-treated or 4H10-treated group. The results indicate that the bloodvessels in tumor tissues are normalized by treatment of antibody 10D6,thereby performing normal functions, to decrease the necrosis of tumortissues.

Example 18 Tumor Metastasis Inhibitory Effect of Anti-Ang2 Antibody

To see the degree of tumor metastasis to near lymph nodes when treatedwith antibody 10D6, referring to the method of Example 14, LLC tumorcells were subcutaneously injected into mice. When the size of tumorsreached 50˜100 mm³, anti-Ang2 antibody 10D6 was injected at theconcentration of 10 mg/kg via an intraperitoneal injection three times aweek and the size of the tumors was measured. After administering theantibody five-times, inguinal lymph nodes were separated, and theobtained tissue sections were immunostained using rabbit anti-LYVE-1antibody (1:200, Millipore) and mouse pan-Cytokeratin antibody (1;200,Abcam).

For comparison, the same experiment was performed to a control grouptreated with Fc of IgG1 and a comparative group treated with antibody4H10 (refer to Example 3).

The obtained results are illustrated in FIG. 17. In FIG. 17, redrepresents lung cancer cells. As shown in FIG. 17, compared toFc-treated or 4H10-treated group, in 10D6-treated group, the size oflymph nodes is decreased (see green region), and the number ofmetastatic lung cancer is decreased (see red region). The resultsindicate that antibody 10D6 inhibits metastasis of lung cancer cells tonear lymph nodes.

Example 19 Effect of Co-Administration of Anti-Ang2 Antibody andAnticancer Agent in Lung Cancer Animal Model

The synergistic effect of co-treatment of antibody 10D6 and cisplatinwas examined.

In particular, referring to Example 14, LLC tumor cells weresubcutaneously injected into mice. When the size of tumors reached50˜100 mm³, anti-Ang2 antibody 10D6 was injected at the concentration of10 mg/kg via an intraperitoneal injection every three days a total offive times. After administering antibody 10D6 twice, 3 mg/kg ofcisplatin (Sigma-Aldrich) was intraperitoneally injected once. The sizeof tumor (V) was calculated using the following formula:

Volume (V)=(width)₂×length/2

For comparison, the same experiment was performed to a control grouptreated with Fc of IgG1 and a comparative group treated with antibody4H10.

The obtained results are illustrated in FIG. 18A. As shown in FIG. 18A,when, antibody 4H10, antibody 10D6, or cisplatin is solely treated, thetumor size is decreased by 29.2%, 38.5%, and 17.6%, respectively,compared to the control; whereas when antibody 4H10 or antibody 10D6 andcisplatin are co-treated, the tumor size is decreased by 42.3%, and66.9%, respectively, compared the control (the numerical values meansthe decline of tumor size compared to that of control). The resultsindicate that the co-treatment of 10D6 and an antibody leads tosignificantly synergistic effect.

In addition, an experiment was performed to examine whether thesynergistic effect obtained by co-administration of 10D6 and cisplatinas described above is due to the increased transporting efficiency ofthe administered cisplatin induced by the vascular normalization bytreatment of 10D6. Cisplatin binds to DNA in a living body and inducescrosslinking, thereby inducing cell death. Rat monoclonal anti-Cisplatinmodified DNA (CP9/19) antibody (1:200, Abcam), which is capable ofbinding to DNA modified by cisplatin in tumor frozen sections, andhamster anti-CD31 antibody (1:200, Millipore), were used forimmunofluorescence staining, and fluorescence images were obtained usingLSM5 confocal microscope (Zeiss). The fluorescence images are shown inFIG. 18B. In addition, to examine necrotic region, the tumor frozensections were stained with H&E according to standard protocol, and then,Eosin+ cell region in tumor was observed. The obtained results areillustrated in FIG. 18C.

As shown in FIG. 18B, larger Cisplatin modified DNA (CP9/19) antibodypositive area is observed in the group co-treated with antibody 10D6 andcisplatin, compared to a group co-treated with Fc and cisplatin or agroup co-treated with antibody 4H10 and cisplatin. In addition, as shownin FIG. 18C, the ratio of Eosin positive area to total tumor area issignificantly increased in the group co-treated with antibody 10D6 andcisplatin, compared to a group co-treated with Fc and cisplatin or agroup co-treated with antibody 4H10 and cisplatin. These resultsindicate that the synergistic effect by co-administration of antibody10D6 and cisplatin is actually caused from the increased efficacy of theco-treated anticancer agent (cisplatin) which is caused by increasedtransporting efficiency of the anticancer agent into tumor tissues byvascular normalization induced by antibody 10D6.

Example 20 Vascular Toxicity of Anti-Ang2 Antibody

The vascular toxicity of antibody 10D6 was tested.

In particular, 5 mg/kg of antibody 10D6 was administered to normal6-week old C57BL/6 mice (Jackson Laboratory) solely or combined with 20ug of Ang2 via an intraperitoneal injection every three days a total of6 times, and then, the weight of the mice was measured. After 20 days,various tissues including thyroid, parathyroid, and adrenal gland wereremoved from the mice, and frozen sections were obtained therefrom. Thefrozen sections were immunostained using hamster anti-CD31 antibody(1:200, Millipore), and the changes were observed. A group administeredwith 20 ug of Ang2 solely was used for comparison.

The obtained results are illustrated with FIGS. 19A and 19B. As shown inFIGS. 19A and 19B, when the normal mice treated with antibody 10D6,neither abnormal weight loss/gain nor morphological abnormality of bloodvessels are observed, indicating that antibody 10D6 has no vasculartoxicity.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. A method of preventing or treating a cancer,comprising administering an anti-Ang2 antibody or an antigen-bindingfragment thereof to a subject in need thereof, wherein the anti-Ang2antibody or the antigen-binding fragment thereof specifically binds toAng2 and forms a complex with a Tie2 receptor and Ang2.
 2. The method ofclaim 1, wherein the anti-Ang2 antibody or an antigen-binding fragmentthereof increases the activity of Tie2 by increasing phosphorylation ofTie2 receptor.
 3. The method of claim 1, wherein the anti-Ang2 antibodyor an antigen-binding fragment thereof increases phosphorylation of atleast one selected from the group consisting of Akt, eNOS, and 42/44. 4.The method of claim 1, wherein the anti-Ang2 antibody or anantigen-binding fragment thereof binds to Q418, P419 or a combination ofQ418 and P419 in human Ang2 of SEQ ID NO: 11; or binds to 2 to 20contiguous amino acid residues of SEQ ID NO: 11 including Q418, P419 ora combination of Q418 and P419.
 5. The method of claim 1, wherein theanti-Ang2 antibody or an antigen-binding fragment thereof comprises: atleast one heavy chain complementarity determining region (CDR) selectedfrom the group consisting of a polypeptide (CDR-H1) including the aminoacid sequence of SEQ ID NO: 1, a polypeptide (CDR-H2) including theamino acid sequence of SEQ ID NO: 2, and a polypeptide (CDR-H3)including the amino acid sequence of SEQ ID NO: 3; at least one lightchain complementarity determining region selected from the groupconsisting of a polypeptide (CDR-L1) including the amino acid sequenceof SEQ ID NO: 4, a polypeptide (CDR-L2) including the amino acidsequence of SEQ ID NO: 5, and a polypeptide (CDR-L3) including the aminoacid sequence of SEQ ID NO: 6; or a combination of said at least oneheavy chain complementarity determining region and said at least onelight chain complementarity determining region.
 6. The method of claim5, wherein the anti-Ang2 antibody or an antigen-binding fragment thereofcomprises: heavy chain complementarity determining regions including apolypeptide (CDR-H1) including the amino acid sequence of SEQ ID NO: 1,a polypeptide (CDR-H2) including the amino acid sequence of SEQ ID NO:2, and a polypeptide (CDR-H3) including the amino acid sequence of SEQID NO: 3, or a heavy chain variable region including the heavy chaincomplementarity determining regions; and light chain complementaritydetermining regions including a polypeptide (CDR-L1) including the aminoacid sequence of SEQ ID NO: 4, a polypeptide (CDR-L2) including theamino acid sequence of SEQ ID NO: 5, and a polypeptide (CDR-L3)including the amino acid sequence of SEQ ID NO: 6, or a light chainvariable region including the light chain complementarity determiningregions.
 7. The method of claim 6, wherein the anti-Ang2 antibody or anantigen-binding fragment thereof comprises a heavy chain variable regionincluding the amino acid sequence of SEQ ID NO: 7 and a light chainvariable region including the amino acid sequence of SEQ ID NO:
 9. 8.The method of claim 1, wherein the anti-Ang2 antibody or anantigen-binding fragment thereof is produced from a hybridoma ofaccession no. KCLRF-BP-00295.
 9. The method of claim 1, furthercomprising a step of administering Ang2 to the subject.
 10. A method ofenhancing an efficacy of an anticancer agent, comprising administeringan anti-Ang2 antibody or an antigen-binding fragment thereof of claim 1to a subject in need thereof.
 11. The method of claim 10, wherein theanti-Ang2 antibody or an antigen-binding fragment thereof increases theactivity of Tie2 by increasing phosphorylation of Tie2 receptor.
 12. Themethod of claim 10, wherein the anti-Ang2 antibody or an antigen-bindingfragment thereof increases phosphorylation of at least one selected fromthe group consisting of Akt, eNOS, and 42/44.
 13. The method of claim10, wherein the anti-Ang2 antibody or an antigen-binding fragmentthereof binds to Q418, P419, or a combination of Q418 and P419 in humanAng2 of SEQ ID NO: 11; or binds to 2 to 20 contiguous amino acidresidues of SEQ ID NO: 11 comprising Q418, P419, or a combination ofQ418 and P419.
 14. The method of claim 10, wherein the anti-Ang2antibody or an antigen-binding fragment thereof comprises: at least oneheavy chain complementarity determining region (CDR) selected from thegroup consisting of a polypeptide (CDR-H1) including the amino acidsequence of SEQ ID NO: 1, a polypeptide (CDR-H2) including the aminoacid sequence of SEQ ID NO: 2, and a polypeptide (CDR-H3) including theamino acid sequence of SEQ ID NO: 3, or a heavy chain variable regionincluding the at least one heavy chain complementarity determiningregion; at least one light chain complementarity determining regionselected from the group consisting of a polypeptide (CDR-L1) includingthe amino acid sequence of SEQ ID NO: 4, a polypeptide (CDR-L2)including the amino acid sequence of SEQ ID NO: 5, and a polypeptide(CDR-L3) including the amino acid sequence of SEQ ID NO: 6, or a lightchain variable region including the at least one light chaincomplementarity determining region; a combination of said at least oneheavy chain complementarity determining region and said at least onelight chain complementarity determining region; or a combination of theheavy chain variable region and the light chain variable region.
 15. Themethod of claim 14, wherein the anti-Ang2 antibody or an antigen-bindingfragment thereof comprises: heavy chain complementarity determiningregions including a polypeptide (CDR-H1) including the amino acidsequence of SEQ ID NO: 1, a polypeptide (CDR-H2) including the aminoacid sequence of SEQ ID NO: 2, and a polypeptide (CDR-H3) including theamino acid sequence of SEQ ID NO: 3, or a heavy chain variable regionincluding the heavy chain complementarity determining regions; and lightchain complementarity determining regions including a polypeptide(CDR-L1) including the amino acid sequence of SEQ ID NO: 4, apolypeptide (CDR-L2) including the amino acid sequence of SEQ ID NO: 5,and a polypeptide (CDR-L3) including the amino acid sequence of SEQ IDNO: 6, or a light chain variable region including the light chaincomplementarity determining regions.
 16. The method of claim 15, whereinthe anti-Ang2 antibody or an antigen-binding fragment thereof comprisesa heavy chain variable region including the amino acid sequence of SEQID NO: 7 and a light chain variable region including the amino acidsequence of SEQ ID NO:
 9. 17. The method of claim 10, wherein theanti-Ang2 antibody or an antigen-binding fragment thereof is producedfrom a hybridoma deposited with accession number KCLRF-BP-00295.
 18. Themethod of claim 10, further comprising administering Ang2 to thesubject.
 19. The method of preventing or treating a cancer of claim 1further comprising administering an anticancer agent to the subject. 20.The method of claim 19, wherein the anticancer agent is at least oneselected from the group consisting of a chemical drug, an antibody, anantisense oligonucleotide, siRNA, shRNA, microRNA, aptamer, a cell fortherapeutic use, and an agent for radiotherapy.
 21. The method of claim19, wherein the anticancer agent is at least one selected from the groupconsisting of cisplatin, carboplatin, oxaliplatin, mechlorethamine,cyclophosphamide, ifosfamide, melphalan, chlorambucil, thiotepa,altretamine, procarbazine, busulfan, carmustine, lomustine, dacarbazine,fluorouracil (5-FU), capecitabine, cytarabine, gemcitabine,methotrexate(MTX), mercaptopurine (6-MP), vinblastine, vincristine,vinorelvine, paclitaxel, docetaxel, etoposide topotecan, irinotecan,dactinomycin, doxorubicin, daunorubicin, mitomycin, bleomycin, imatinib,trastuzumab, cetuximab, gefitinib, erlotinib, bevacizumab, sunitinib,sorafenib, cabozantinib, pazopanib, regorafenib, vandetanib,ziv-afilibercept, prednisone, and 6-thioguanine (6-TG).
 22. The methodof claim 19, further comprising administering Ang2 to the subject.